Method of identifying susceptibility to angiotensin converting enzyme inhibto- and vasopeptidase-inhibitor-associated angioedema

ABSTRACT

Deficiencies in certain physiological pathways are linked with ACE or vasopeptidase inhibitor associated angioedema. Additionally, detection and/or measurement of dipeptidyl peptidase IV (DPP IV) enzyme activity and aminopeptidase P (APP) enzyme activity is a predictor of this risk. The present invention provides biological markers, diagnostic tests, and pharmaceutical indications that are useful in the diagnosis and treatment of angioedema and in the marketing and safety of certain medications. This ability can be important for the treatment of a subject that is in need of or are taking an angiotensin-converting enzyme (ACE) inhibitor and/or a vasopeptidase inhibitor (combined ACE and neutral endopeptidase (NEP) inhibitor), which are commonly used in the treatment of hypertension (high blood pressure), diabetes, and cardiac and renal diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

The present patent application is based on and claims priority to U.S.Provisional Application Ser. No. 60/244,524, entitled “BiologicalMarkers and Diagnostic Tests for Angiotensin Converting Enzyme Inhibitorand Vasopeptidase Inhibitor Associated Angioedema”, which was filed Oct.31, 2000 and is incorporated herein by reference.

GRANT STATEMENT

This invention was made with federal grant money under NIH grantsHL56963, GM 07569 and 5M01 RR-00095. Thus, the United States Governmenthas certain rights in the present invention.

TECHNICAL FIELD

The present invention relates generally to screening tests to determinewhich patients are at risk for developing angioedema associated withinhibitors of angiotensin converting enzyme (ACE) and/or combined ACEand neutral endopeptidase (NEP) inhibitors (a combined ACE/NEP inhibitoris referred to herein as a “vasopeptidase inhibitor”). Moreparticularly, the present invention relates to an association betweendipeptidyl peptidase IV (DPP IV) and aminopeptidase P (APP) enzymaticactivity and ACE and vasopeptidase inhibitor-related angioedema. Thepresent invention also provides screening tests and kits to identify asubject who is at risk for ACE and vasopeptidase inhibitor-associatedangioedema.

Abbreviations ACE angiotensin converting enzyme ACEI angiotensinconverting enzyme inhibitor AGT angiotensinogen ANP atrial natriuteticpeptide APP aminopeptidase P DPP IV dipeptidyl peptidase IV HTNhypertensive NCBI National Center for Biotechnology Information NEPneutral endopeptidase NLM National Library of Medicine NTN normotensiveOMIM Online Mendelian Inheritance in Man RAS renin-angiotensin system

BACKGROUND ART

Administration of angiotensin-converting enzyme (ACE) inhibitors iscommon medical practice for the treatment of a variety of diseaseconditions, including: cardiac and renal diseases, diabetes, andhypertension (high blood pressure). Several combined ACE and neutralendopeptidase (NEP) inhibitors are presently under investigation or areawaiting regulatory approval for the treatment of the aforementioneddisease conditions. However, the administration of an ACE and/or avasopeptidase inhibitor (referred to herein as an ACE/vasopeptidaseinhibitor) is contraindicated for subjects with a history of angioedemadue to the potential severity of this side effect, which can be sosevere as to result in death. Approximately 0.1% to 1.0% of thepopulation receiving an ACE inhibitor is predicted to be susceptible todeveloping at least one episode of angioedema during treatment. Thispercentage might be even higher, especially for subjects taking avasopeptidase inhibitor. Also, these inhibitors are often administeredover long periods of time because the illnesses that they treat areoften chronic conditions. This could increase the chances of a subjectdeveloping angioedema over a course of treatment.

Angioedema is an uncommon, but serious, side effect of ACE andvasopeptidase inhibitors. Currently, it is not possible to accuratelypredict which subjects are at risk to develop angioedema when taking anACE or vasopeptidase inhibitor; however it is known that approximately0.1% to 1.0% or more of the subjects receiving an ACE or vasopeptidaseinhibitor will develop angioedema as a side effect. The variation insusceptibility to vasopeptidase-associated angioedema depends, in part,on the subgroup of the population that is analyzed. For example, AfricanAmericans are particularly susceptible to ACE inhibitor associatedangioedema.

In patients who develop angioedema while taking one of thesemedications, it is difficult to determine if the angioedemic conditionarose in response to the medication or due to some other occurrence. Forexample, certain allergic reactions can result in angioedema. Thecurrent standard in practice is to employ a treatment other than anACE/vasopeptidase inhibitor, if a patient has a known history ofangioedema, or to halt treatment with ACE/vasopeptidase inhibitors if apatient presents with symptoms of angioedema or it is learnedafter-the-fact that the patient has a history of angioedema. Mostpractitioners, however, consider these alternative therapies to be lesseffective in treating the original condition than ACE/vasopeptidaseinhibitor therapy.

What is needed, therefore, are tests, assays, and biological markers foridentifying patients that are at increased risk for developingangioedema related to treatment with ACE/vasopeptidase inhibitors, ascompared to the general population or a matched population. Such assayswould allow the continued use of ACE/vasopeptidase inhibitors insubjects that have a reduced susceptibility to angioedema and therational regulation of their use in susceptible subjects. The presentinvention solves these and other problems, in part by providingbiological markers and diagnostic tests and kits that are preferablyemployed early on in treatment, thereby averting complications.

SUMMARY OF THE INVENTION

A method of identifying a subject that is susceptible to developing anangioedemic condition during a course of treatment comprisingadministering one of an ACE inhibitor and a vasopeptidase inhibitor isdisclosed. In a preferred embodiment, the method comprises (a) providinga biological sample obtained from a subject; (b) determining adipeptidyl peptidase IV activity in the biological sample; and (c)comparing a dipeptidyl peptidase IV activity in the biological sample toa standard dipeptidyl peptidase IV activity, wherein a 10% or morereduction in the sample activity compared to the standard indicates thatthe subject is susceptible to developing an angioedema during a courseof treatment comprising administering one of an ACE inhibitor and avasopeptidase inhibitor. Preferably, the vasopeptidase inhibitor is anangiotensin-converting enzyme inhibitor or a neutral endopeptidaseinhibitor. It is also preferable that a 20% or more reduction in thesample activity compared to the standard indicates that the subject issusceptible and that the subject is a human.

A method of identifying a subject that is susceptible to developing anangioedemic condition during a course of treatment comprisingadministering one of an ACE inhibitor and a vasopeptidase inhibitor isdisclosed. In a preferred embodiment, the method comprises: (a)providing a biological sample obtained from a subject; (b) determiningan aminopeptidase P activity in the biological sample; and (c) comparingan aminopeptidase P activity activity in the biological sample to astandard aminopeptidase P activity, wherein a 10% or more reduction inthe sample activity compared to the standard indicates that the subjectis susceptible to developing an angioedema during a course of treatmentcomprising administering one of an ACE inhibitor and a vasopeptidaseinhibitor. Preferably, the vasopeptidase inhibitor is anangiotensin-converting enzyme inhibitor or a neutral endopeptidaseinhibitor. It is also preferable that a 20% or more reduction in thesample activity compared to the standard indicates that the subject issusceptible and that the subject is a human.

A method of determining contraindication for administration of one of anACE inhibitor and a vasopeptidase inhibitor to an individual isdisclosed. In a preferred embodiment, the method comprises: (a)providing a biological sample obtained from a subject; (b) determining adipeptidyl peptidase IV activity in the biological sample; and (c)comparing a dipeptidyl peptidase IV activity in the biological sample toa standard dipeptidyl peptidase IV activity, wherein administration ofthe vasopeptidase inhibitor is contraindicated when the dipeptidylpeptidase IV activity in the biological sample is outside the standarddipeptidyl peptidase IV activity range.

A method of determining contraindication for administration of one of anACE inhibitor and a vasopeptidase inhibitor to an individual isdisclosed. In a preferred embodiment, the method comprises: (a)providing a biological sample obtained from a subject; (b) determiningan aminopeptidase P activity in the biological sample; and (c) comparingan aminopeptidase P activity in the biological sample to a standardaminopeptidase P activity, wherein administration of the vasopeptidaseinhibitor is contraindicated when the aminopeptidase P activity in thebiological sample is outside the standard aminopeptidase P activityrange.

A method of screening an individual for compatibility with anadministration of one of an ACE inhibitor and a vasopeptidase inhibitoris disclosed. In a preferred embodiment, the method comprises: (a)providing a biological sample obtained from a subject; (b) determining adipeptidyl peptidase IV activity in the biological sample; and (c)comparing a dipeptidyl peptidase IV activity in the biological sample toa standard dipeptidyl peptidase IV activity range, whereinadministration of the vasopeptidase inhibitor is contraindicated whenthe sample activity is outside the standard dipeptidyl peptidase IVactivity range, and wherein administration of the vasopeptidaseinhibitor is indicated when the sample activity is either within orabove the standard dipeptidyl peptidase IV activity range. Preferably,the vasopeptidase inhibitor is an angiotensin-converting enzymeinhibitor or a neutral endopeptidase inhibitor.

A method of screening an individual for compatibility with anadministration of one of an ACE inhibitor and a vasopeptidase inhibitoris disclosed. In a preferred embodiment, the method comprises (a)providing a biological sample obtained from a subject; (b) determiningan aminopeptidase P activity in the biological sample; and (c) comparingan aminopeptidase P activity in the biological sample to a standardaminopeptidase P activity range, wherein administration of avasopeptidase inhibitor is contraindicated when the sample activity isbelow the standard aminopeptidase P activity range, and whereinadministration of the vasopeptidase inhibitor is indicated when thesample activity is either equal to or above the standard aminopeptidaseP activity range. Preferably, the vasopeptidase inhibitor is anangiotensin-converting enzyme inhibitor or a neutral endopeptidaseinhibitor.

A kit for identifying a subject at risk for angioedema during a courseof treatment comprising administering one of an ACE inhibitor and avasopeptidase inhibitor is disclosed. In a preferred embodiment, the kitcomprises: (a) a substrate of a dipeptidyl peptidase IV enzyme; (b) abuffer; (c) a reaction stop solution; and (d) a set of instructionscomprising information on a standard dipeptidyl peptidase IV activityrange. Preferably, the article of manufacture further comprises acalibration solution for calibration of the reaction and the substrateis Gly-Pro-p-nitroanilide.

A kit for identifying a subject at risk for angioedema during a courseof treatment comprising administering one of an ACE inhibitor and avasopeptidase inhibitor is disclosed. In a preferred embodiment, the kitcomprises: (a) an aminopeptidase P enzyme substrate; (b) a dilutionbuffer; (c) a reaction stop solution; (d) a revelation buffer; and (e) aset of instructions comprising information on a standard aminopeptidaseP activity range. Preferably, the article of manufacture furthercomprises a calibration solution for calibration of the reaction and thesubstrate is the peptide Arg-Pro-Pro.

A kit for identifying a subject at risk for angioedema during a courseof treatment comprising administering one of an ACE inhibitor and avasopeptidase inhibitor is disclosed. In a preferred embodiment, the kitcomprises (a) a vasopeptidase inhibitor; and (b) a packaging materialcomprising information that the vasopeptidase inhibitor iscontraindicated for individuals with a serum dipeptidyl peptidase IVenzyme activity outside a standard dipeptidyl peptidase IV activityrange.

A kit for identifying a subject at risk for angioedema during a courseof treatment comprising administering one of an ACE inhibitor and avasopeptidase inhibitor is disclosed. In a preferred embodiment, the kitcomprises (a) a vasopeptidase inhibitor; and (b) a packaging materialcomprising information that the vasopeptidase inhibitor iscontraindicated for individuals with a serum aminopeptidase P enzymeactivity outside a standard aminopeptidase P activity range.

Another kit is disclosed and in a preferred embodiment comprises avasopeptidase inhibitor and a packaging material, wherein the packagingmaterial includes information that the vasopeptidase inhibitor iscontraindicated for individuals with a dipeptidyl peptidase IV enzymeactivity below a normal range or is indicated for individuals with adipeptidyl peptidase IV enzyme activity within a normal range.

Another kit is disclosed and in a preferred embodiment comprises avasopeptidase inhibitor and a packaging material, wherein the packagingmaterial includes information that the vasopeptidase inhibitor iscontraindicated for individuals with an aminopeptidase P enzyme activitybelow a normal range or is indicated for individuals with anaminopeptidase P enzyme activity within a normal range.

A method of marketing a vasopeptidase inhibitor is disclosed and in apreferred embodiment, the method comprises providing information about adiagnostic test adapted to identify a subject that is susceptible toangioedema as a result of taking the vasopeptidase inhibitor during acourse of treatment comprising administering one of an ACE inhibitor anda vasopeptidase inhibitor. Preferably, the vasopeptidase inhibitor is anangiotensin-converting enzyme inhibitor, the diagnostic test comprisesdetecting an activity of a dipeptidyl peptidase IV enzyme or anaminopeptidase P enzyme in a biological sample from the subject, and thesubject is a human. It is also preferable that the vasopeptidaseinhibitor is a neutral endopeptidase inhibitor that the diagnostic testincludes detecting an activity of a dipeptidyl peptidase IV enzyme or anaminopeptidase P enzyme in a biological sample from the subject, and thesubject is a human.

Accordingly, it is an object of the present invention to provide a novelmethod and article for identifying a subject that is susceptible todeveloping an angioedemic condition during a course of treatmentcomprising administering one of an ACE inhibitor and a vasopeptidaseinhibitor. This and other objects are achieved in whole or in part bythe present invention.

An object of the invention having been stated hereinabove, other objectswill be evident as the description proceeds, when taken in connectionwith the accompanying Drawings and Laboratory Examples as best describedhereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting an overview of selected portions of therenin-angiotensin system (RAS) and a Substance P metabolic pathway.

FIG. 2 is a diagram depicting an overview of angiotensin-convertingenzyme (ACE) inhibitor and neutral endopeptidase (NEP) inhibitor actionon the systems/pathways described in FIG. 1.

FIG. 3 is a diagram depicting the catalysis of angiotensin I toangiotensin II by ACE and includes the amino acid residue sequence (SEQID NOS:1 and 2) of each species and the major position for enzymaticcleavage of the angiotensin I amino acid residue chain.

FIG. 4A is a diagram depicting the catalysis of bradykinin (SEQ ID NO:3)into inactive metabolites by ACE and NEP (arrows depict the sites ofenzymatic cleavage; cleavage sites of the dipeptidyl peptidase IV (DPPIV) and aminopeptidase P (APP) pathways for the degradation ofbradykinin into inactive metabolites are indicated by dashed arrows).

FIG. 4B is a diagram depicting the catalysis of substance P (SEQ IDNO:4) by ACE and NEP. The arrows depict the sites of enzymatic cleavage(a cleavage site of the DPP IV pathway for the degradation of substanceP into inactive metabolites is indicated by a dashed arrow).

FIG. 5 is a plot depicting DDP IV activity (innanomoles/milliliter/minute or nM/ml/min) in a control population(Control), a population with ACE inhibitor (ACEI) associated angioedema(ACEI-associated), and a population treated with an ACE inhibitor butwithout angioedema (non-ACEI).

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is an amino acid sequence of a peptide fragment ofangiotensin I.

SEQ ID NO: 2 is an amino acid sequence of a peptide fragment ofangiotensin II.

SEQ ID NO: 3 is an amino acid sequence of a peptide fragment ofbradykin.

SEQ ID NO: 4 is an amino acid sequence of a peptide fragment ofsubstance P.

SEQ ID NO: 5 is a nucleotide sequence encoding human dipeptidylpeptidase IV.

SEQ ID NO: 6 is an amino acid sequence of human dipeptidyl peptidase IV.

SEQ ID NO: 7 is a nucleotide sequence encoding a soluble form of humanaminopeptidase P.

SEQ ID NO: 8 is an amino acid sequence of a soluble form of humanaminopeptidase P.

SEQ ID NO: 9 is a nucleotide sequence encoding a membrane-bound form ofhuman amino peptidase P.

SEQ ID NO: 10 is an amino acid sequence of a membrane-bound form ofhuman amino peptidase P.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides biological markers, diagnostic tests,clinical assays, and articles of manufacture (such as kits useful in thetests and assays) for identifying an increased risk for developingACE/vasopeptidase inhibitor-associated angioedema in a subject. Thepresent invention also provides information for an appropriate course oftreatment for individuals taking ACE/vasopeptidase inhibitormedications. The articles and methods of the present invention can alsobe employed to identify a subject that has a reduced risk for developingACE/vasopeptidase inhibitor associated angioedema.

For example, by employing the articles of manufacture and methods of thepresent invention, a physician can determine whether or not treatmentwith an ACE/vasopeptidase inhibitor is advisable based upon a risk thatthe subject might develop angioedema. Likewise, a physician caring for asubject that has been started on an ACE/vasopeptidase inhibitor canlearn that the subject has a history of one or more events of angioedemaunrelated to ACE or vasopeptidase inhibitor treatment. The physician canemploy the methods of the present invention to determine if the subjectis susceptible to ACE/vasopeptidase associated angioedema. If not, or ifthe risk is low, then the physician can continue treatment with theACE/vasopeptidase inhibitor. If the subject is determined to besusceptible to developing ACE/vasopeptidase inhibitor angioedema, thephysician can discontinue treatment with the ACE/vasopeptidaseinhibitor, or can optionally select an alternative mode of treatment.

In another situation, a subject might present with angioedema whilebeing treated with an ACE/vasopeptidase inhibitor. In this case, thephysician typically would discontinue treatment with theACE/vasopeptidase inhibitor until the angioedemic condition is resolved.The methods and articles of the present invention can be employed todetermine whether the angioedema resulted from the administration of theACE/vasopeptidase inhibitor or if it is likely to be due to anothercause, whether defined or undefined. If the determination by the presentinvention is that the cause is not due to administration of theACE/vasopeptidase inhibitor, then the physician can restart treatmentwith an ACE/vasopeptidase inhibitor. If the determination by the presentinvention is that the cause is due to administration of theACE/vasopeptidase inhibitor (or likely due), then the physician canselect an alternative mode of treatment (ACE/vasopeptidase inhibitorsare contraindicated in this latter situation).

In another example, during the research, development, and/or manufactureof an ACE/vasopeptidase inhibitor compounds, a pharmaceutical company orother entity can employ the methods and articles of the presentinvention to evaluate the safety of the compounds. Alternatively, theentity might desire to screen test populations in order to identifysubjects that are at increased risk of developing serious side effects,such as angioedema, associated with the administration of thecompound(s) being tested. This can make the testing period more safe forthe subjects being evaluated. Moreover, the present invention can reducethe possibility of negative consequences from the sale ofACE/vasopeptidase inhibitors because, after a assessment performed withthe methods and articles of the present invention, the ACE/vasopeptidaseinhibitors can be contraindicated for the populations that are most atrisk.

In addition to the market for treatment of humans, ACE and/orvasopeptidase inhibitors are used to treat similar illness in pets,livestock and show animals and the methods and compositions of thepresent invention are generally applicable to these other mammals. Theoccurrence of angioedema as a side effect, even in a relatively smallfraction of the population being treated with ACE/vasopeptidaseinhibitors, has serious consequences in the marketability of these drugsand the availability of these drugs to the approximately 99% of thetreated population that does not develop angioedema.

Animals so treated can be warm-blooded vertebrates, for instance,mammals and birds. More particularly, the animal can be selected fromthe group consisting of rodent, swine, bird, ruminant, and primate. Evenmore particularly, the animal can be selected from the group consistingof a mouse, a rat, a pig, a guinea pig, poultry, an emu, an ostrich, agoat, a cow, a sheep, and a rabbit. Most particularly, the animal can bea primate, such as an ape, a monkey, a lemur, a tarsier, a marmoset, ora human.

Thus, provided is the treatment of mammals such as humans, as well asthose mammals of importance due to being endangered (such as Siberiantigers), of economical importance (animals raised on farms forconsumption by humans) and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans (such ascats and dogs), swine (pigs, hogs, and wild boars), ruminants (such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also provided is the treatment of birds, including the treatmentof those kinds of birds that are endangered, kept in zoos, as well asfowl, and more particularly domesticated fowl, e.g., poultry, such asturkeys, chickens, ducks, geese, guinea fowl, and the like, as they arealso of economical importance to humans. Thus, provided is the treatmentof livestock, including, but not limited to, domesticated swine (pigsand hogs), ruminants, horses, poultry, and the like.

I. Definitions

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

The term “about”, as used herein when referring to a measurable valuesuch as an amount of activity, weight, time, dose, etc. is meant toencompass variations of ±2%, even more preferably ±1%, and still morepreferably ±0.1% from the specified amount, as such variations areappropriate to perform the disclosed method.

As used herein, the terms “biological marker” and “biomarker” are usedinterchangeably and carry the meaning as understood by one of ordinaryskill in the art. The term specifically encompasses a testable ormeasurable indicator that can be linked or associated with a phenotypeor trait. The indicator can be enzymatic, genetic, biochemical,physiological, or other form as known in the art.

As used herein, the term “ACE/vasopeptidase inhibitor” means aninhibitor of ACE and/or an inhibitor of vasopeptidase. Thus, anACE/vasopeptidase inhibitor can comprise an ACE inhibitor and/or acombined ACE and NEP inhibitor.

As used herein, the term “ACE inhibitor” means an inhibitor ofangiotensin converting enzyme (ACE).

As used herein, the term “health care provider” is known in the art andspecifically includes a physician, a person with authority to prescribea medication (whether directly or indirectly), and a veterinarian. Incertain embodiments, a health care provider includes an individual thatprovides a medication without prescription, such as in providing anover-the-counter medication.

As used herein, the terms “identifying subjects” and “diagnosing” areused interchangeably with regard to the detection of a “predisposition”,“increased propensity”, “risk”, “increased risk”, and the like. Theterms specifically encompass identifying the propensity for a subject todevelop ACE/vasopeptidase inhibitor associated angioedema.

As used herein, the terms “standard”, “normal range”, “control range”,and “clinical range” have normal meanings as known in the art. As usedherein, these terms do not apply to DPP IV or APP enzyme activity inpopulations that have ACE/vasopeptidase inhibitor associated angioedemaat the time of detection or measurement. The terms “subject range” or“experimental range” and the like are descriptive of enzyme activityranges in subjects or patients with ACE/vasopeptidase inhibitorassociated angioedema (acute or in the patient history). One of ordinaryskill in the art can determine the clinical ranges for a givenpopulation and numerous clinical ranges and standards are known in theart for a variety of enzyme activities.

As used herein, the terms “vasopeptidase enzyme” and “vasopeptidase” areused interchangeably and include, but are not limited to,angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP).Other vasopeptidases will be known to those with skill in the art.

As used herein, the term “vasopeptidase inhibitor” includes, but is notlimited to, compounds that inhibit both ACE and neutral endopeptidase(NEP).

As used herein, the term “ACE/vasopeptidase inhibitor” means an ACEinhibitor and/or a vasopeptidase inhibitor.

As used herein, the term “contraindicated” means a symptom or conditionthat makes a treatment, procedure, or administration of a medicationinadvisable.

As used herein, the terms “detecting” and “detect” are usedinterchangeably and mean qualitative and/or quantitative determinations,including measuring an amount of enzyme activity in terms of units ofactivity or units activity per unit time, and the like.

As used herein, the terms “standard dipeptidyl peptidase IV activity”and “standard aminopeptidase P activity” mean an activity thatrepresents an average measurement of the APP and DPP IV activities of anumber of individuals. The activities can be measured by employingactivity assays such as those disclosed herein. A standard activity canbe employed as a benchmark against which an activity observed in asample is gauged.

As used herein, the term “angioedemic condition” means a condition in asubject comprising at least the onset of symptoms consistent with aclinical diagnosis of angioedema. An angioedemic condition can comprisesymptoms and effects peripherally associated with angioedema or symptomsand effects arising as a result of the onset or presence of angioedema.

The term “subject” as used herein refers to any invertebrate orvertebrate species. The methods of the present invention areparticularly useful in the treatment of warm-blooded vertebrates. Thus,in a preferred embodiment, the invention concerns mammals and birds.

II. General Considerations

Angiotensin-converting enzyme (ACE) inhibitors and vasopeptidaseinhibitors are indicated for the treatment of hypertension, congestiveheart failure, diabetic neuropathy, coronary artery disease, and certainother conditions. In addition, considerable research efforts are ongoingto further improve treatment of these conditions with ACE andvasopeptidase inhibitors and to identify new inhibitors. These aremedically important drugs with large markets for the treatment of humansand other mammals.

The present invention provides biological markers, diagnostic tests,assays, kits, and pharmaceutical indications which are useful foridentifying individuals susceptible to developing angioedema associatedwith treatment by an angiotensin converting enzyme (ACE) inhibitor or avasopeptidase inhibitor. The markers, tests, assays, kits andindications described herein, are generally applicable to humans andother mammals.

It will be understood that the methods and articles of the presentinvention can be employed to identify subjects or individuals that arecompatible with administration of ACE/vasopeptidase inhibitors. Forthese subjects, ACE/vasopeptidase inhibitor treatment might be indicateddepending on their need for such treatment as determined by one ofordinary skill in the art.

II.A. Angiotensin Converting Enzyme

Angiotensin-converting enzyme (ACE) catalyzes the cleavage ofangiotensin I into angiotesin II, which has an activity of raising bloodpressure (see FIG. 1). ACE and NEP catalyze the degradation ofbradykinin and substance P into inactive metabolites. NEP also catalyzesthe degradation of atrial natriutetic peptide (ANP) into inactivemetabolites. In contrast to angiotesin II, bradykinin and ANP have anactivity of lowering blood pressure. Therefore, the use oradministration of an ACE/vasopeptidase inhibitor generally results in areduction in blood pressure because these inhibitors reduce angiotesinII production and increase bradykinin and/or ANP concentrations byinhibiting their degradation into inactive metabolites (see FIG. 2).Included in the many additional applications of ACE inhibitors are thetreatment of cardiac diseases, renal diseases, and diabetes.Vasopeptidase inhibitors are also under investigation for use in theseconditions and are awaiting regulatory approval. The clinicaleffectiveness of these inhibitors might result from influences onmultiple physiological pathways, however, and the present invention isin no way bound by theory or mechanism.

The ACE enzymatic pathway is the primary pathway for angiotesin IIformation and bradykinin degradation (see FIG. 3). Alternative pathwayshave been identified for the degradation of both bradykinin andsubstance P, however (see FIGS. 4A and 4B). These pathways comprise thedegradation of bradykinin by the aminopeptidase P (APP) and dipeptidylpeptidase IV (DPP IV) enzymes, and the degradation of substance P by DPPIV. In general, the contribution of the alternative DPP IV and APPpathways could, but not necessarily, increase during ACE/vasopeptidaseinhibition for individuals that are at a reduced risk of angioedema(“non-ACEI”) even in comparison to normotensives (“Control”, see FIG.5). On the other hand, individuals with increased angioedema risk(“ACEI-associated”) show a reduction alternative pathway activity (forexample, DPP IV).

II.B. Angiotensin Converting Enzyme and Vasopeptidase Inhibitors

As noted, ACE acts on converting angiotensin I to angiotesin II.Angiotensin II increases blood pressure and is considered a main causeof essential hypertension. A variety of studies have been directed tosubstances inhibiting ACE actions, primarily addressing the suppressionof a rise in blood pressure.

Therapeutic vasodepressors such as CAPTOPRIL™ andD-2-methyl-3-mercaptopropanoyl-L-proline have been synthesized as ACEinhibitors. Additional ACE inhibitors available commercially includeENALAPRIL™, ENALAPRILAT™, QUINAPRIL™, RAMIPRIL™, CILAZAPRIL™, DELAPRIL™,FOSENOPRIL™, ZOFENOPRIL™, INDOLAPRIL™, LISINOPRIL™, PERINDOPRIL™,SPIRAPRIL™, PENTOPRIL™, PIVOPRIL™, and known pharmaceutically acceptablesalts thereof. From foodstuff, peptides having ACE inhibiting activitieshave been separated through enzymatic hydrolysis of casein (JapaneseLaid-Open Patent Publication Nos. 62-270533, 64-5497, 64-83096) andsoybean protein (Japanese Laid-Open Patent Publication Nos. 3-1671981).

Synthetic ACE inhibitors exhibit strong activities, and can exhibitadverse effects (such as angioedema). ACE inhibitory peptides derivedfrom casein or soybean protein have been developed with expectation oflow toxicity and high safety, even though they exhibit low activities.Recent studies, therefore, have been focused on separating ACEinhibitors from foodstuff materials and manufacturing them on a largescale by chemical synthetic methods.

An ACE inhibitor derived from food protein was first reported in 1979 byOshima et al. (Oshima et al., (1979) Biochim. Biophys. Acta 556: 128).Since then over 40 ACE inhibitory peptides have been disclosed to date(see, e.g., Ariyoshi, (1993) Trends Food Sci. Tech., May, 1993, p. 139).A number of ACE inhibitory peptides have been derived from foodstuffsuch as sour milk (Nakamura et al., (1995) J. Dairy Sci. 78: 777), tunatissue (Kohama et al., (1988) Biochem. Biophys. Res. Comm. 155(1): 332),sardine muscle (Matsuda et al., (1992) Nippon Nogeigaku Kaishi 66(11):1645), oyster protein (Matsumoto et al., (1994) Nippon Shokuhin KogyoGakkaishi 41(9): 589), Ficus carica (Maruyama et al., (1989) Agric.Biol. Chem. 53(10): 2763), and rice (Muramoto & Kawamora, (1991) FoodInd. 34(11): 18). Furthermore, numerous patent applications have beenfiled in relation with ACE inhibitory peptides, including synthesizedinhibitors as well as those isolated from natural products See e.g.,U.S. Pat. Nos. 5,449,661; 5,071,955; 4,692,459; 4,585,758; 4,512,979;4,191,753; 3,832,337; and European Patent No. EP174162.

II.C. Angioedema

It has been observed that treatment with ACE/vasopeptidase inhibitors isassociated with the development of angioedema in a small percentage ofindividuals. The affected population accounts for approximately 0.1% toapproximately 1.0% of patients receiving treatment withACE/vasopeptidase inhibitors and appears to be more prevalent amongAfrican Americans than Caucasian Americans.

In general, angioedema is a swelling of tissue and especially affectsthe lips and other parts of the mouth, throat, larynx, eyelids,genitals, hands, and feet. Angioedema of the mouth, tongue and larynxcan be life threatening especially when severe swelling makes breathingdifficult.

The present inventor has discovered that deficiencies in the dipeptidylpeptidase IV (DPP IV) and aminopeptidase P (APP) enzymatic pathways arerelated to vasopeptidase inhibitor associated angioedema. For example,the present inventor discovered that DPP IV and/or APP activity isreduced in individuals with ACE associated angioedema compared toactivity in patients with hypertension who have been treated with an ACEinhibitor but have not had angioedema.

III. Biological Markers

The present invention provides biological markers (also known asbiomarkers) for identifying subjects or individuals with asusceptibility to ACE/vasopeptidase inhibitor associated angioedema. Forexample, as described herein, a low DPP IV serum enzymatic activity isassociated with an increased risk that an individual will developangioedema if an ACE/vasopeptidase inhibitor is administered. In anotherexample, as described herein, a low APP serum enzymatic activity isassociated with an increased risk that an individual will developangioedema if an ACE/vasopeptidase inhibitor is administered. Thus,biological markers specifically encompasses a testable or measurableindicator that can be linked or associated with a phenotype or trait.The indicator can be enzymatic, genetic, biochemical, physiological, orother form as known in the art. Summarily, a biological marker or abiomarker demonstrates a correlation between a first condition and asecond condition.

In one aspect of the present invention, dipeptidyl peptidase IV (DPP IV)activity is a biological marker for ACE/vasopeptidase inhibitorassociated angioedema. In another aspect of the present invention,aminopeptidase P (APP) activity is a biological marker forACE/vasopeptidase inhibitor associated angioedema. In general, theactivity of either enzyme is preferably detected in a biological sampleof the subject, and more preferably a serum sample. In certainembodiments, other useful biological samples include, but are notlimited to: tissue, biopsy, interstitial fluid, feces, urine, wholeblood, and epithelium. The biological samples can be collected andprocessed according to methods known in the art for measuring enzymaticactivity (or with adaptations as would be apparent from the disclosurehereof).

In certain embodiments, the level of enzymatic activity can be measuredqualitatively and, in other embodiments, the level of enzymatic activitycan be measured quantitatively. In certain embodiments for theevaluation of ACE/vasopeptidase inhibitor associated angioedema, DPP IVactivity can be measured and analyzed; in other embodiments APP activitycan be measured and analyzed; and in yet other embodiments, both DPP IVand APP activities can be measured and analyzed. The same is true forqualitative detection of the biological markers. Several assays aredescribed in the Examples. In general, a qualitative assay can include areaction substrate that is placed in the biological sample and reactedwith the DPP IV and/or APP enzyme present in the sample. The reactionsubstrate can change colors, for example, if the examined activity istoo low/high by a relative amount, and a color change can indicatedetection of activity. The reaction substrate can be compared to asimilar substrate preparation reacted with a control or standard. Incertain embodiments, DPP IV and/or APP enzymatic activity in abiological sample obtained from a subject can be measured in vitro andin other embodiments, it can be measured in vivo. In general, themeasured activity is inversely proportional to the risk forACE/vasopeptidase inhibitor associated angioedema. Laboratory Example 1demonstrates the use of DPP IV as a biological marker in the context ofthe present invention.

In certain embodiments, a health care professional can test a subjectfor risk for developing an ACE/vasopeptidase inhibitor associatedangioedema by a method comprising: detecting or measuring a serum DPP IVand/or APP activity; administering the ACE/vasopeptidase inhibitor for atime sufficient to inhibit ACE and/or NEP activity; and then detectingor measuring the serum DPP IV and/or APP activity again, for example,after a period of time has lapsed.

In certain aspects of this embodiment, an increase in DPP IV and/or APPactivity indicates that the subject has a low risk for developingACE/vasopeptidase inhibitor associated angioedema. In certain otherembodiments, a decrease in DPP IV and/or APP activity indicates that thesubject has a high risk for developing ACE/vasopeptidase inhibitorassociated angioedema. In yet other aspects of this embodiment, a DPP IVand/or APP activity that does not significantly change indicates thatthe subject has an intermediate to high risk for developingACE/vasopeptidase inhibitor associated angioedema.

A subject's risk of developing an angioedemic condition can be analyzedat any time, for example, when considering administering anACE/vasopeptidase inhibitor to the subject or after the administrationhas commenced. Also, the diagnostic tests described herein (which canrely on one or more biological markers) can be employed to evaluate thecause of angioedema in a patient that is currently taking anACE/vasopeptidase inhibitor.

IV. ACE Inhibitors and Vasopeptidase Inhibitors

ACE inhibitors can differ in the chemical structure of their activemoieties, in potency, in bioavailability, in plasma half-life, in routeof elimination, in their distribution and affinity for tissue-bound ACE,and in whether they are administered as prodrugs. The same can be truefor vasopeptidase inhibitors. Those of ordinary skill in the artrecognize that the side effects of ACE inhibitors can be divided intothose that are class specific and those that relate to specific agents.ACE inhibitors decrease systemic vascular resistance without increasingheart rate and they promote natriuresis. ACE inhibitors have provedeffective in the treatment of hypertension. ACE inhibitors also decreasemortality in congestive heart failure and left ventricular dysfunctionafter myocardial infarction, and they delay the progression of diabeticnephropathy.

Certain examples of known and commercially available ACE inhibitors arelisted in Table 1. This is not meant to be an exhaustive list, butmerely exemplary of certain ACE inhibitors that can be employed intreating subjects in need of treatment therewith. An example of avasopeptidase inhibitor in development includes omapatrilat (brand nameVANLEV™ by Bristol-Meyers Squibb).

TABLE 1 Marketed ACE Inhibitors Company Compound Name (Maker of Brand(Generic Drug) Brand Name Name) Captopril CAPOTEN Enalapril VASOTECMerck Lisinopril ZESTRIL Zeneca Lisinopril PRINIVIL Merck BenazeprilLOTENSIN Novartis Quinapril ACCUPRIL Parke-Davis Ramipril ALTACE MonarchTrandolapril MAVIK Knoll (Roussel Uclaf) Moexipril UNIVASE SchwartzFosinopril MONOPRIL BMS Perindep ACESRI Solva

V. ACE/Vasopeptidase Inhibitor-Associated Angioedema

ACE inhibitors have been shown to reduce mortality in patients withcongestive heart failure, diabetic nephropathy, and coronary arterydisease. In addition to ACE inhibitor-produced effects in reducingangiotensin II production, evidence from both animal studies and humanstudies indicate that cardioprotective effects of ACE inhibitors derivein part through potentiation of the effects of bradykinin (Gainer etal., (1998) New Engl. J. Med. 339: 1285-92, incorporated herein byreference). Another group of drugs have been identified with combinedACE/NEP inhibitory effects (these drugs are included in the meaning ofthe term “vasopeptidase inhibitors”), that block degradation ofbradykinin and substance P through two pathways and also block thedegradation of atrial natriutetic peptide (ANP). These combined ACE/NEPinhibitor medications appear to be particularly effect in lowering bloodpressure in hypertensive African Americans.

While it is not the inventor's desire to be bound to theory ormechanism, it is postulated that some aspect of bradykinin and/orsubstance P plays a role in potentiating angioedema (Emanueli et al.,(1998) Hypertension 31:1299-1304; Kim et al., (2000) J. Pharm. Exp.Ther. 292: 295-298; Ersahin et al., (1997) J. Cardiovasc. Pharm. 30:96-101; Blais et al., (1999) Immunopharmacology 43: 293-302; Blais etal., (1999) Peptides 20: 421-430; Damas et al., (1996) N-S Arch.Pharmacol. 354: 662-669, all of which are incorporated herein byreference). For example, an over accumulation of bradykinin and/orsubstance P might help potentiate ACE/vasopeptidase inhibitor associatedangioedema. Thus, using this example, it is postulated by the inventorthat inhibition of bradykinin and/or substance P breakdown by ACE orcombined ACE/NEP inhibitor action has beneficial effects up to a point;however, certain individuals appear to have an inability to clear anexcessive accumulation of bradykinin and/or substance P leading to anincreased risk of developing angioedema.

The risk of ACE inhibitor-associated angioedema is increased in AfricanAmericans compared to Caucasians, suggesting that genetic factors canmodulate risk of angioedema (Brown et al., (1996) Clin. Pharmacol. Ther.60: 8-13, incorporated herein by reference). Also, the inventor hasobserved that there is a large number of transplant recipients among thepatients with angioedema. Again, without being bound to any theory ormechanism, the inventor hypothesizes that cyclosporin A, which iscommonly used to treat transplant patients and also inhibits serum DPPIV activity (Scharpe et al. (1990) Clin. Chem. 36: 984), results inACE/vasopeptidase inhibitor associated angioedema in transplantrecipients. Thus, a genetic and/or an acquired defect in theaminopeptidase P and/or dipeptidyl peptidase IV pathways, which serve asalternative pathways for the degradation of bradykinin and substance P,are described herein to predispose patients to the development of ACEinhibitor or vasopeptidase inhibitor angioedema.

VI. Peptide, Polypeptide and Polynucleotide Components of the PresentInvention

A variety of biological information including nucleotide and peptidesequence information is available from public databases provided, forexample, by the National Center for Biotechnology Information (NCBI)located at the United States National Library of Medicine (NLM). TheNCBI is located on the world wide web at the URL“http://www.ncbi.nlm.nih.gov/” and the NLM is located on the world wideweb at the URL “http://www.nlm.nih.gov/”. The NCBI website providesaccess to a number of scientific database resources including: GenBank,PubMed, Genomes, LocusLink, Online Mendelian Inheritance in Man (OMIM),Proteins, and Structures. A common interface to the polypeptide andpolynucleotide databases is referred to as Entrez which can be accessedfrom the NCBI website on the World Wide Web at URL“http://www.ncbi.nlm.nih.gov/Entrez/” or through the LocusLink website.

The following subsections disclose a plurality of molecules that canform an element of the present invention. This discussion is not meantto be an inclusive list of molecules that can form a component of thepresent invention. The following subsections are included to provideadditional detail regarding components of the present invention, as wellas to help illustrate how the various molecules relate to one another invivo.

VI.A. Angiotensin I and Angiotensin II

The following summary is available in the NCBI LocusLink database:

The human AGT gene product, pre-angiotensinogen, is expressed in theliver and is cleaved by the enzyme renin in response to lowered bloodpressure. The resulting product, angiotensin I is then cleaved byangiotensin converting enzyme (ACE) to generate the physiologicallyactive enzyme [sic, peptide] angiotensin II. Human pre-angiotensinogenis encoded by two mRNAs that differ only in the length of the3′-untranslated region due to postulated use of two polyadenylationsites. There may also be alternative initiation codons (nucleotides40-42 and 67-69). AGT is involved in maintaining blood pressure and inthe pathogenesis of essential hypertension and preeclampsia.

The Homo sapiens Official Gene Symbol and Name is: AGT: angiotensinogen.In a preferred embodiment of the present invention, angiotensin Icomprises the amino acid sequence of SEQ ID NO: 1. The hormoneangiotesin II is recognized as one of the most potent vasopressor agentsthat produces hypertension in mammals. The action of the enzyme renin onthe plasma protein substrate angiotensinogen results in the productionof an inactive decapeptide, angiotensin 1, which upon conversion by thenon-selective angiotensin converting enzyme (ACE) provides angiotesinII, the active hormone. See e.g., Regoli et al., (1974) Pharm. Rev. 26:69.

Angiotensin II causes vasoconstriction and stimulates aldosteronesecretion (from the adrenal gland) that results in a rise of both bloodvolume and pressure. Inhibitors of angiotesin II are therefore useful intreating hypertension, congestive heart failure, renal insufficiencyassociated with diabetic or hypertensive nephropathy, and glaucoma. Seee.g., Garrison et al., in The Pharmacological Basis of Therapeutics, 8thEdition, (Gilman, Goodman, Rall, Nies, and Taylor, eds), Pergamon Press,New York, 1990: p. 761-762; and Dzau, (1991) New Engl. J. Med. 324:1124-1130.

Angiotensin II also can act on other organs such as the brain(Fitzsimmons, (1980) Rev. Physiol. Biochem. Pharmacol. 87: 117).Antagonists of angiotesin II are therefore useful in enhancing cognitiveperformance in patients affected by conditions such as age associatedmental impairment or Alzheimer's disease, and in treating cognitivedisorders such as anxiety. See e.g., Dennes et al., (1992) Brit. J.Pharmacol. 105: 88; and Barnes et al., (1991) FASEB J., 5: 678.

In addition, angiotesin II acts on a variety of glandular tissuesincluding the kidney, liver, and ovaries. Antagonists of angiotesin IIare useful in treating conditions, disorders, or diseases of thesetissues associated with excessive or unregulated angiotesin II activity.Antagonists of angiotesin II are also useful in treating kidney damagedue to non-steroidal antiinflammatory agents.

Angiotensin II has a role in regulation of the rate of cell growth anddifferentiation. Inhibitors of angiotesin II are therefore useful intreating disorders marked by excessive cell proliferation such asrestenosis. See, e.g., Naftilan et al., (1989) J. Clin. Invest. 83:1419, Kauffman et al., (1991) Life Sci. 49: 223-228, and Jackson et al.,(1988) Nature 335: 437. Angiotensin II is formed in the human bodythrough proteolysis of angiotensin I (Ang I) primarily through theaction of angiotensin-converting enzyme (see FIG. 1). In a preferredembodiment of the present invention, angiotesin II comprises the aminoacid sequence of SEQ ID NO: 2.

VI.B. Bradykinin

Bradykinin is a nonapeptide generated as a result of the activity ofkallikreins, a group of proteolytic enzymes present in most tissues andbody fluids, on kininogens. Once released, kinins produce manyphysiological responses, including pain and hyperanalgesia bystimulating C- and A-fibers in the periphery. There is also considerableevidence that kinins contribute to the inflammatory response.

Bradykinin, and its physiologically important related peptides kallidin(Lys-bradykinin) and Met-Lys-bradykinin, exhibit physiological actionswhich qualify them as mediators of inflammatory reactions, hypotensivestates, and pain. Bradykinin is overproduced in pathological conditionssuch as septic shock, anaphylaxis, rhinitis, asthma, inflammatory boweldisease, and certain other conditions including acute pancreatitis,post-gastrectomy dumping syndrome, carcinoid syndrome, migraine, andangioneurotic edema. The production of bradykinin from the plasmaresults in pain at the site of the pathological condition, and theoverproduction intensifies the pain directly or via bradykinin-inducedactivation of the arachidonic acid pathway which produces prostaglandinsand leukotrienes, the more distal and actual mediators of inflammation.

In addition to its analgesic and proinflammatory effects, bradykinin isa vasodilator. Because of its ability to lower blood pressure,bradykinin has been implicated in the pathogenesis of several shocksyndromes, particularly septic or endotoxic shock. Bradykinin is also apotent bronchoconstrictor in animals and asthmatic subjects and it hasbeen implicated as a contributor to the pathogenesis of airwayinflammatory conditions such as allergic asthma and rhinitis. In apreferred embodiment of the present invention, bradykinin comprises theamino acid sequence of SEQ ID NO: 3

Summarily, bradykinin increases vascular permeability, dilates bloodvessels, increases blood flow, contracts non-vascular smooth muscle(e.g., bronchial), stimulates pain, and lowers blood pressure(hypotensive). These are also cardinal signs of inflammation. Bradykininis formed by the cleavage of kininogen by the enzyme kallikrein, and israpidly cleared in the mammalian body by cleavage into inactivemetabolites (see FIG. 1) primarily by angiotensin-converting enzyme(ACE) and neutral endopeptidase (NEP).

VI.C. Substance P

Substance P is a naturally occurring undecapeptide belonging to thetachykinin family of peptides, the latter being so-named because oftheir prompt stimulatory action on smooth muscle tissue. More specially,substance P is a pharmaceutically active neuropeptide that is producedin mammals (having originally been isolated from gut) and possesses acharacteristic amino acid sequence that is illustrated by Veber et al.in U.S. Pat. No. 4,680,283. The wide involvement of substance P andother tachykinins in the pathophysiology of numerous diseases has beenamply demonstrated in the art. For instance, substance P has recentlybeen shown to be involved in the transmission of pain or migraine, aswell as in central nervous system disorders such as anxiety andschizophrenia, in respiratory and inflammatory diseases such as asthmaand rheumatoid arthritis, respectively, and in gastrointestinaldisorders and diseases of GI tract, like ulcerative colitis and Crohn'sdiseases, etc. It is also reported that the tachykinin antagonists areuseful for the treatment of allergic conditions, immunoregulation,vasodilation, bronchospasm, reflex or neuronal control of the visceraand senile dementia of the Alzheimer's type, emesis, sunburn andHelicobacter pylori infection.

Substance P is similar to bradykinin in function in that substance Pstimulates: smooth muscle contraction, inflammation, and blood vesseldilation. Substance P also functions in neurotransmission, histaminerelease, and activation of the immune system. Substance P is synthesizedin neurons and, similar to bradykinin, is degraded into inactivemetabolites by ACE and NEP. In a preferred embodiment of the presentinvention, substance P comprises the amino acid sequence of SEQ ID NO:4.

VI.D. Dipeptidyl Peptidase IV (DPP IV)

Dipeptidyl peptidase IV (DPPIV) is a serine protease that cleavesN-terminal dipeptides from a peptide chain containing, preferably, aproline residue in the penultimate position. Although the biologicalrole of DPP-IV in mammalian systems has not been completely established,it is believed to play an important role in neuropeptide metabolism,T-cell activation, attachment of cancer cells to the endothelium, andthe entry of HIV into lymphoid cells.

Various types of dipeptidyl peptidase IV have been purified and theenzymological properties have been revealed. For example, the dipeptidylpeptidase IV is isolated from rat liver (Hopsu-Havu & Glenner, (1966)Histochem. 7: 197-201), swine kidney (Barth et al., (1974) Acta Biol.Med. Chem. 32:157-174), small intestine (Svensson et al., (1978) Eur. J.Biochem. 90: 489-498), liver (Fukasawa et al., (1981) Biochim. Biophys.Acta 657: 179-189), human submaxillary gland (Ova et al., (1972)Biochim. Biophys. Acta 258: 591-599), sheep kidney (Yoshimoto & Walter,(1977) Biochim. Biophys. Acta, 485: 391-401; Yoshimoto et al., (1978) J.Biol. Chem. 253: 3708-3716) or microorganisms (Fukusawa & Harada, (1981)Arch. Biochem. Biophys. 210: 230-237; Yoshimoto & Tsuru, (1982) Biochem.91:1899-1906).

The DPP IV enzyme is a serine exopeptidase that cleaves X-prolinedipeptides from the N-terminus of polypeptides. It is an intrinsicmembrane glycoprotein anchored into the cell membrane by its N-terminalend. Soluble forms of DPP IV are also known including those in the serum(Struyf et al., (1999) J. Immunol. 162: 4903-4909, incorporated hereinby reference). High levels of DPP IV enzyme are found in thebrush-border membranes of the kidney proximal tubule and of the smallintestine, but several other tissues also express the enzyme. DPP IVcleaves bradykinin and substance P into inactive (or reduced activity)metabolites as shown in FIGS. 4A and 4B. Table 2 discloses additionalembodiments of DPP IV.

TABLE 2 Embodiments of GenBank Sequences for DPP4 (DPP4 is generallyreferred to herein as DPP IV) Nucleotide Type Protein AH005372 gAAB60646 U13710 g AAB60646 U13711 g AAB60646 U13712 g AAB60646 U13713 gAAB60646 U13714 g AAB60646 U13715 g AAB60646 U13716 g AAB60646 U13717 gAAB60646 U13718 g AAB60646 U13719 g AAB60646 U13720 g AAB60646 U13721 gAAB60646 U13722 g AAB60646 U13723 g AAB60646 U13724 g AAB60646 U13725 gAAB60646 U13726 g AAB60646 U13727 g AAB60646 U13728 g AAB60646 U13729 gAAB60646 U13730 g AAB60646 U13731 g AAB60646 U13732 g AAB60646 U13733 gAAB60646 U13734 g AAB60646 U13735 g AAB60646 M74777 m AAA51943 M80536 mAAA52308 X60708 m CAA43118

VI.E. Aminopeptidase P

Aminopeptidase P is known to cleave the N-terminal amino acid frompeptides that have a prolyl residue in the second position (Orawski etal., (1987) Mol. Cell. Biochem. 75: 123-132; Simmons & Orawski, (1992)J. Biol. Chem. 267: 4897-4903; Yoshimoto et al., (1994) Arch. Biochem.Biophys. 311: 28-34). It has been suggested that membrane-boundaminopeptidase P has an important role in vivo in the pulmonarydegradation of bradykinin (Ryan et al., (1994) J. Pharmacol. Exper.Thera. 269: 941-947; Ryan, (1989) Am. J. Physiol. 257: L53-L60; Orawski(1987) Mol. Cell. Biochem. 75: 123-132; Orawski, (1989) Adv. Exp. Med.Biol. 2478: 355-364; Simmons & Orawski, (1992) J. Biol. Chem. 267,4897-4903; Kitamura, (1995) Br. J. Pharmacol. 114: 6-7; Baker (1991)Cir. Shock 33: 37-47; Pesquero et al., (1992) J. Hyperten. 10:1471-1478; Pasquero et al., (1992) J. Hyperten. 10: 1479-1484) bycleaving its Arg¹-Pro² bond. It has also been suggested that otherpeptidases could also play a role in bradykinin degradation (Orawski etal., (1989) Adv. Exp. Med. Biol. 2478: 355-364).

Several embodiments of the aminopeptidase P enzyme are useful in thepresent invention. Examples of useful embodiments are described herein,but are not meant to limit the present invention.

VI.E.1. Aminopeptidase P (Aminopeptidase 1, Soluble)

One embodiment is the APP referred to by Homo sapiens Official GeneSymbol and Name: XPNPEP1: X-prolyl aminopeptidase (aminopeptidase P) 1,soluble. Table 3 presents an additional embodiment of APP.

TABLE 3 Certain GenBank Sequences for Aminopeptidase P1 Nucleotide TypeProtein AF195530 m AAF97866

VI.E.2. Aminopeptidase P (Aminopeptidase 2, Membrane-Bound)

Another useful embodiment is the APP referred to, in the NCBI LocusLinkdatabase, by Homo sapiens Official Gene Symbol and Name XPNPEP2:X-prolyl aminopeptidase (aminopeptidase P) 2, membrane-bound. Table 4presents additional embodiments of APP2.

TABLE 4 Certain GenBank Sequences for Aminopeptidase P2 Nucleotide TypeProtein AL023653 g CAA19220 U90724 m AAB96394

VII. Dipeptidyl Peptidase IV Activity Assay

The present invention also comprises an assay for dipeptidyl peptidaseIV. In a preferred embodiment, the steps for performing the assay are asfollows. Initially, samples comprising 0, 25, 50 and 100 units (e.g.,nM/ml) of p-nitroaniline are prepared for generating a standard curve.p-nitroaniline is a known substrate for DPP IV. The standard curve isgenerated by determining the absorbance of the standard solutions ofp-nitroaniline at 405 nm and are plotted on a graph as concentrationversus absorbance.

To perform a DPP IV assay on a sample obtained from a subject (e.g., ahuman serum sample), 20 μl of sample is incubated with 10 μl of 2 mMGly-Pro-p-nitroanilide in 170 μM 0.1 M Tris-HCl for 1 hour. The reactionis stopped by adding 800 μl sodium acetate (1 M, pH 4.5) and theabsorbance is measured at 405 nm. The concentration of p-nitroanilineformed per ml per min is then calculated by employing a standard curve.The activity and/or presence of DPP IV in the sample can be determinedby comparing the observed activity with a standard activity.

VIII. Aminopeptidase P Activity Assay

The present invention also comprises an aminopeptidase P activity assay.In a preferred embodiment, the steps for performing an APP assay are asfollows. First, a calibration curve is prepared by monitoringfluorescence emission at 310 and 445 nm (excitation and emissionwavelengths, respectively) from a range of concentrations of 1-arginine(0-5 mM).

Next, a sample is provided (e.g. a human serum sample). 20 μl of thesample is incubated at 37° C. with 180 μl HEPES buffer containing 5.6 mMArg-Pro-Pro, yielding a final concentration of Arg-Pro-Pro of 0.5 mM.Arg-Pro-Pro is a known substrate for APP. After an incubation period oftwo hours, the reaction is stopped by adding 800 μl of cold, anhydrousethanol to the reaction mixture. The mixture is then centrifuged at2000×g at 4° C. for 15 minutes. The supernatant is decanted andincubated at room temperature with 3 ml of a revelation buffer. APPactivity is calculated as nmoles arginine released per min per ml ofserum sample.

IX. Applications of the Present Invention

The present invention can be employed in a range of applications.Preferably, the present invention is employed in a situation in which aphysician is contemplating a course of treatment comprising an ACEinhibitor, a vasopeptidase inhibitor and combinations thereof. In thissituation, the present invention can be employed to minimize the riskthat a patient might develop an angioedemic condition.

The present invention can be employed, for example, to identify asubject that is susceptible to developing an angioedemic conditionduring a course of treatment which comprises administering an ACEinhibitor, a vasopeptidase inhibitor or, as is commonly the case, acombination thereof. The present invention can also be employed todetermine if administration of an ACE inhibitor, a vasopeptidaseinhibitor, or a combination thereof, is contraindicated for a subject.In a related application, the present invention can be employed in amethod of screening a subject for compatibility with administration of avasopeptidase. Additionally, the present invention can be marketed inthe form of diagnostic kits, which a physician or a researcher canemploy to identify a subject at risk for angioedema during a course oftreatment which comprises administering an ACE inhibitor, avasopeptidase inhibitor or, as is commonly the case, a combinationthereof. These are just a few of the range of applications in which thepresent invention can be employed. These applications are described morefully herein below and in the Examples that follow.

IX.A. Method of Identifying a Subject That is Susceptible to Developingan Angioedemic Condition During a Course of Treatment

An aspect of the present invention is the observation that there is alink between DPP IV and/or APP activity, ACE and/or vasopeptidaseinhibitors and the onset of an angioedemic condition. Thus, when asubject is undergoing a course of treatment comprising administering anACE inhibitor, a vasopeptidase inhibitor or a combination thereof, it ispreferable to determine the activity of DPP IV and/or APP in a samplederived from the subject. Depressed DPP IV and/or APP activity levelsindicate that the subject is at risk for developing an angioedemiccondition as a result of the course of treatment.

In a preferred embodiment of this application of the present invention,a biological sample is initially provided by a subject. Preferably, thesample is a serum sample. A sample can be acquired from a subject byemploying standard techniques, and will be dependent, in part, on thenature of the sample. For example, when the sample comprises a sample ofthe subject's blood, standard phlebotomic methods can be employed toacquire the sample, which can be further processed as required (e.g.isolating a serum component of sample).

Following sample acquisition and preparation (if required), a standardDPP IV and/or APP activity is determined. A standard DPP IV and/or APPactivity can be determined by calculating DPP IV and/or APP activity ina control group of subjects. The number of subjects can vary, butpreferably, the number of subjects is sufficiently large as to permitthe identification of significant activity measurement. Similarly, thegenetic qualities of the subjects can vary or can be held constant, atthe preference of the researcher. This calculated activity can beemployed as a standard (i.e. a standard DPP IV and/or APP activity),against which a subject's determined DPP IV and/or APP activity isgauged.

Subsequently, DPP IV and/or APP activity present in the sample can bedetermined. Both a standard DPP IV and/or APP, as well as DPP IV and/orAPP activity present in a sample, can be measured by employing, forexample, the activity assays disclosed herein, particularly in sectionVII (DPP IV activity) and in section VIII (APP activity).

When a value is determined for both a standard DPP IV and/or APPactivity and DPP IV and/or APP activity present in a sample, the twovalues can be compared. If DPP IV and/or APP activity in the sample isfound to be less than the activity of the control group (i.e., astandard activity) by about 10% or more, the subject is at risk for anangioedemic condition, should ACE and/or vasopeptidase inhibitor therapybe started or continued. Thus, ACE and/or vasopeptidase inhibitortherapy is contraindicated for subjects in which the DPP IV and/or APPactivity of a sample is found to be less than the activity of thecontrol group (i.e., a standard activity) by about 10% or more. On theother hand, ACE and/or vasopeptidase inhibitor therapy can be toleratedand/or indicated for subjects in which the DPP IV and/or APP activity ofa sample is found to be within about 10% or less of the activity of thecontrol group (i.e. a standard activity).

A 20% or more reduction in the DPP IV and/or APP activity in thebiological sample, as compared to the standard DPP IV and/or APPactivity also indicates that the subject is susceptible to developing anangioedema during a course of treatment comprising administering one ofan ACE inhibitor and a vasopeptidase inhibitor. Additionally, a 30% ormore reduction in the DPP IV and/or APP activity in the biologicalsample, as compared to the standard DPP IV and/or APP activity indicatesthat the subject is susceptible to developing an angioedema during acourse of treatment comprising administering one of an ACE inhibitor anda vasopeptidase inhibitor.

IX.B. Method of Determining Contraindication for Administration of aVasopeptidase Inhibitor, an ACE Inhibitor and Combinations Thereof

In another aspect of the present invention, a vasopeptidate inhibitor,an ACE inhibitor and combinations thereof can be contraindicated if DPPIV and/or APP activity is found to fall outside the range of normalactivities and/or amounts. APP and DPP IV activities can be determinedby employing the assays disclosed in the present invention. In apreferred method of determining contraindication for administration ofan ACE inhibitor or a vasopeptidase inhibitor to an individual, abiological sample obtained from a subject is initially provided.Preferably, the biological sample comprises serum and is obtained from ahuman subject, although the method can also be performed in the contextof an organism other than a human and a sample can comprise a materialother than serum.

Next, a standard DPP IV activity and/or APP activity can be determinedand can be plotted to generate a standard curve. The standard DPP IVactivity and/or APP activity can be determined by measuring a DPP IVand/or APP activity from a number of representative subjects. A standardDPP IV activity and/or APP activity measurement can serve as a benchmarkagainst which a DPP IV activity and/or APP activity observed in a sampleis measured.

Following providing (and preparing, if desired) a biological sample, aDPP IV activity and/or a APP activity for the biological sample can bedetermined. The DPP IV and/or APP activities can be determined asdisclosed herein, and are preferably performed under the same conditionsas were employed in generating the standard activity (i.e. the standardcurve).

Observed DPP IV activity and/or APP activity in the biological samplecan then be compared to the standard DPP IV activity and or APPactivity. If the comparison indicates that DPP IV and/or APP activity inthe biological sample is below the normal range, administration of anACE or a vasopeptidase inhibitor can be contraindicated.Contraindication of administration of an ACE inhibitor or avasopeptidase inhibitor can impart the beneficial effect of decreasingor eliminating the chance that a subject will develop an angioedemiccondition.

IX.C. A Kit For Identifying a Subject at Risk for Angioedema During aCourse of Treatment Comprising Administering an ACE Inhibitor, aVasopeptidase Inhibitor or a Combination Thereof

In another aspect of the present invention, a kit for identifying asubject at risk for angioedema during a course of treatment comprisingadministering an ACE inhibitor, a vasopeptidase inhibitor or acombination thereof is disclosed. Such a kit can be employed by aphysician, laboratory researcher or other person desiring to identify anindividual at risk for developing an angioedemic condition. In apreferred embodiment, the kit comprises a substrate for a DPP IV enzyme.Such a substrate preferably comprises gly-pro-p-nitroanilide; however,other substrates can be employed.

A kit of the present invention also preferable comprises a buffer, whichcan function to maintain pH and other conditions in an optimal range fora DPP assay. Any buffer adapted to maintain a set of desired conditions(e.g., pH, tonicity, etc) can be employed in a kit. A reaction stopsolution is also preferably included in the kit. The reaction stopsolution can be added to a reaction mixture in order to halt any DPPIV-catalyzed reaction occurring in the reaction mixture at a desiredtime point.

Additionally, a kit preferably comprises a set of instructionscomprising information on a range of dipeptidyl peptidase IV activity ina control population. The information contained in such a set ofinstructions can advise a physician or researcher (or any person) who isemploying the kit on the question of how to compare a DPP IV activityobserved in a sample with a standard DPP IV activity. In other words, aset of instructions can advise the user of the kit how to interpret theresults of a test performed by employing the kit. A set of instructionscan also comprise step-by-step directions on how a user can employ thevarious components of the kit to generate an observed DPP IV activityfrom a sample. Thus, such a set of instructions can comprise informationon volumes of solutions to be added, incubation time periods,wavelengths to monitor (if any) and other parameters of a DPP IV assay.

In practice, if an observed DPP IV activity falls within a rangespecified in a set of instructions, administering an ACE inhibitor, avasopeptidase inhibitor or a combination thereof can be administered toa subject with the knowledge that the risk of the subject developing anangioedemic condition is minimal. Thus, such a kit can be employed toidentify a subject at risk for developing an angioedemic conditionbefore a course of treatment comprising administering a vasopeptidaseinhibitor and/or an ACE inhibitor.

In another embodiment of a kit for identifying a subject at risk forangioedema during a course of treatment comprising administering an ACEinhibitor, a vasopeptidase inhibitor or a combination thereof, the kitcomprises an APP substrate. A suitable APP substrate can comprise, forexample, a peptide sequence comprising Arg-Pro-Pro. A dilution buffercan also be included and can be used to dilute a substrate solution orother concentrated solution supplied with the kit or derived from asample acquired from a subject. A reaction stop solution can also beincluded, as well as a revelation buffer. The revelation buffer canassist in maintaining conditions under which APP activity in a samplecan be determined. For example, if a colorimetric assay is employed, arevelation buffer can be employed to develop a degree of color.Alternatively, if a spectrophotometric assay is employed, the revelationbuffer can be employed to maintain conditions under which a detectablereaction product can remain in a detectable state (i.e. undegraded).

Additionally, a set of instructions comprising information on a range ofAPP activity in a control population can be provided with a kit of thepresent invention. As described above in the context of DPP IV, if anobserved APP activity falls within a range specified in a set ofinstructions, administering an ACE inhibitor, a vasopeptidase inhibitoror a combination thereof can be administered to a subject with theknowledge that the risk of the subject developing an angioedemiccondition is minimal. Thus, such a kit can be employed to identify asubject at risk for developing an angioedemic condition before a courseof treatment comprising administering a vasopeptidase inhibitor and/oran ACE inhibitor.

In yet another embodiment, a kit for identifying a subject at risk forangioedema during a course of treatment comprising administering an ACEinhibitor, a vasopeptidase inhibitor or a combination comprises an ACEinhibitor and/or a vasopeptidase inhibitor; and a packaging materialcomprising information that the vasopeptidase inhibitor iscontraindicated for individuals with a serum DPP IV enzyme activityand/or a serum APP enzyme activity below a normal range, which can bespecified in the packaging material.

IX.D. Method of Marketing a Vasopeptidase and/or an ACE Inhibitor

A method of marketing a vasopeptidase and/or an ACE inhibitor is alsodisclosed. In one embodiment, information about a diagnostic testadapted to identify a subject that is susceptible to angioedema as aresult of taking the vasopeptidase inhibitor during a course oftreatment comprising administering an ACE inhibitor, a vasopeptidaseinhibitor, or a combination thereof is provided. When it is known that agiven subject might be at risk for developing an angioedemic condition,this information can comprise an element of a marketing approach. Inthis way, a vasopeptidase and/or ACE inhibitor can be marketed toindividuals who can tolerate these inhibitors, while subjects that mightbe susceptible to developing an angioedemic condition as a result of acourse of treatment comprising these inhibitors can be advised of thisrisk.

This information can be presented to a consumer, whether the consumer isa physician or a subject, at the time an inhibitor is purchased.Alternatively, the information can be presented to a consumer at a pointprior to purchase. This method of marketing can be advantageous becauseit is not only a marketing tool, but can also decrease the risk of asubject developing an angioedemic condition.

X. Illustrative Examples of Preferred Embodiments

This section of the present disclosure provides illustrative examples ofthe application of the present invention. The Illustrative Examples,therefore, provide additional guidance in the application of the presentinvention. These illustrative examples resemble medical case studies,since the present invention is preferably suited to therapeuticapplication (and therefore of particular benefit to physicians), inaddition to being a valuable research tool. The Illustrative Examplesare ordered similarly; first, facts of the case are presented, andsubsequently, several outcomes are presented. These outcomes describetreatments a physician can recommend. In the Illustrative Examples, thephysician in the examples arrives at his or her recommendation as aresult of employing the present invention. In other words, the physicianorders a test, which involves various aspects of the present invention(i.e. a determination of DPP IV activity, APP activity, etc). Thephysician then evaluates the results of the test and recommends a courseof treatment. Thus, the alternative outcomes presented in theIllustrative Examples are based on the results of the test or testsordered by the physician. The Illustrative Examples, therefore, serve todemonstrate how the present invention can be employed in a clinicalsetting.

ILLUSTRATIVE EXAMPLE 1

A 55-year-old African American woman smoker with diabetic nephropathypresents to clinic with poorly controlled hypertension. She is takinghydrochlorothiazide alone for treatment of her hypertension. Because ofthe patient's diabetic nephropathy the patient's physician determinesthat an ACE inhibitor is the drug of choice for treatment of her highblood pressure. However, based on demographic factors, the physiciancalculates that the patient's risk of ACE inhibitor-associatedangioedema is high (1:400). (One of ordinary skill in the art is able tocalculate an individual's risk based upon the scientific literature andthe race of the patients.) He therefore draws blood for measurement ofDPP IV activity and APP activity prior to starting her on an ACEinhibitor.

Outcome A of Illustrative Example 1

The patient's DPP IV and APP activities are found to be normal and shecarries no genetic alleles associated with decreased activity. On thisbasis, the physician calculates that the patient's risk of angioedema islower than predicted by demographics and starts her on an ACE inhibitor.

Outcome B of Example 1

The patient is found to have decreased DPP IV activity. On this basisher calculated risk of angioedema is unacceptably high and the physicianchooses an alternative therapy.

ILLUSTRATIVE EXAMPLE 2

A 64-year-old African American man with dilated cardiomyopathy and ahistory of congestive heart failure presents to the emergency room withswelling of his lips and oropharynx. On examination he is noted to bestridorous and he is intubated to protect his airway. He is givenintravenous corticosteroids and histamine H₁ and H₂ antagonists. Priorto admission he was taking the diuretic furosemide, the ACE inhibitorlisinopril, and the aldosterone receptor antagonist spironolactone. Hehas taken the ACE inhibitor for at least four years and has never hadany previous episode of angioedema. Five days prior to admission he wasstarted on the antibiotic ciprofloxacin for a urinary tract infection.It was not clear to the patient's physician whether his angioedema wasrelated to his use of an ACE inhibitor. Given the proven benefit of ACEinhibitors as treatment in patients with left ventricular dysfunction,the physician desired to continue therapy, if possible. The physiciandraws blood samples for measurement of C₁ esterase inhibitor activity,APP and DPP IV activity, as well as a sample for extraction and analysisof DNA markers and sequences.

Outcome A of Illustrative Example 2

C₁ esterase inhibitor activity is found to be normal, excluding C₁esterase inhibitor deficiency associated hereditary angioedema. However,DPP IV activity is found to be below the normal range. It is determinedthat it is not safe to restart the patient's ACE inhibitor, since therisk of angioedema is high.

Outcome B of Illustrative Example 2

The C₁ esterase inhibitor activity is found to be normal, excluding C₁esterase inhibitor associated hereditary angioedema. The DPP IV activityis found to be below the normal range. The physician determines thattreatment with the ACE inhibitor is still the best possible mode oftreatment, once the angioedema is resolved, and the physician wants todetermine if biomarkers and biochemical indicators (e.g., DPP IVactivity) reveal that the angioedema was an isolated episode possiblyrelated to some other exposure. Thus, the DPP IV activity is measuredagain in about 2 weeks or more after the first measurement (or after theangioedema has resolved).

Outcome B1 of Illustrative Example 2

The DPP IV activity found to remain depressed even after the angioedemahas resolved. The physician determines that the risk of a recurrentepisode of angioedema is high and orders that the ACE/vasopeptidaseinhibitor treatment should not be restarted.

Outcome B2 of Illustrative Example 2

The DPP IV activity found to increase sufficiently after the angioedemahas resolved or returns to normal, such that the physician determinesthat the angioedema was related to an isolated acquired influence. Thephysician determines that the patient's episode of angioedema is likelyrelated to concurrent ciprofloxacin administration and that the risk ofa recurrent episode of angioedema is low. The ACE or vasopeptidaseinhibitor treatment is restarted at the original dose level or,alternatively, at a lower dose than the original dose of ACE orvasopeptidase inhibitor.

ILLUSTRATIVE EXAMPLE 3

A physician determines that a patient is in need of treatment with anACE/vasopeptidase inhibitor. A blood sample is drawn from the patientand is processed to obtain a serum sample. The DPP IV and/or APPactivity is determined for the individual. The patient is started on theinhibitor(s). The DPP IV and/or APP enzyme activity is checkedperiodically to determine the risk for angioedema and to determine ifthe risk is changing. The period between tests can be any periodselected by the physician. In certain examples the period is about oneweek, in certain examples the period is about six months and in certainexamples the period varies from test to test.

ILLUSTRATIVE EXAMPLE 4

Example 4 is the same as Example 3, except that the patient developsangioedema during the course of treatment with the inhibitor(s).Treatment with the inhibitor(s) is suspended until the angioedema isresolved and until the DPP IV and/or APP enzyme activity is found to beat a safe level(s) to resume treatment with the inhibitor(s).

LABORATORY EXAMPLES

The following Laboratory Examples have been included to illustratepreferred modes of the invention. Certain aspects of the followingLaboratory Examples are described in terms of techniques and proceduresfound or contemplated by the present inventors to work well in thepractice of the invention. These Laboratory Examples are exemplifiedthrough the use of standard laboratory practices of the inventors. Inlight of the present disclosure and the general level of skill in theart, those of skill will appreciate that the following LaboratoryExamples are intended to be exemplary only and that numerous changes,modifications and alterations can be employed without departing from thespirit and scope of the invention.

Laboratory Example 1

One use of DPP IV enzyme activity as a biological marker is demonstratedin FIG. 5. In this example, DPP IV activity is in a range of about 28 toabout 42 nM/ml/min in a control group. The control group comprisessubjects that have received an ACE or vasopeptidase inhibitor and do nothave angioedema (they are normotensive). Thus, 28 to 42 nM/ml/min isconsidered to be the normal range or control range for this particularpopulation, in this example. DPP IV activity in a group of hypertensivesubjects who have received an ACE inhibitor, but were free fromangioedema, is in a range above the normotensive control group in thisexperiment. Thus, above 28 and preferably above 40 nM/ml/min isconsidered to be the normal range or control range for this particulargroup (for example, 40 to 50 nM/ml/min; for another example 40 to morethan 40 nM/ml/min). A group receiving an ACE inhibitor and presentingwith acute angioedema has reduced DPP IV enzymatic activity. The subjectrange is between 18 and 27 nM/ml/min, in this example. Thus, this groupshows a reduction in the average and the median DPP IV activity comparedto the hypertensive group. There is a significant difference in theranges of DPP IV activity between these groups and the significance isgreater than or equal to a 95% confidence interval.

Referring now to Table 5, Column A (NTN) is normotensive controls.Column B (HTN) is hypertensive controls (received ACE inhibitor at sometime). Column C is a subject group with acute angioedema and receivingACE inhibitor. Values that are outside a “range” can be outside of the95% confidence interval, for example.

TABLE 5 RESULTS OF A CLINICAL TRIAL X Labels A B C D E X Labels NTN HTNACEI AE ACEI AE non-ACEI AE X Y Y Y Y Y Number of 21 10 5 7 2 valuesMinimum 24.80 28.08 23.97 19.68 41.25 25% Percentile 34.21 30.28 31.61Median 38.06 35.41 24.61 35.57 42.06 75% Percentile 42.80 39.17 43.15Maximum 51.59 39.75 28.38 43.57 42.87 Mean 37.76 34.59 25.32 35.12 42.06Std. Deviation 6.300 4.243 1.774 8.511 1.146 Std. Error 1.375 1.3420.7935 3.217 0.8100 Lower 95% CI 34.90 31.55 23.12 27.25 31.77 Upper 95%CI 40.63 37.62 27.53 42.99 52.35

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It will be understood that various details of the invention may bechanged without departing from the scope of the invention. Moreover, itis not the inventor's desire to be bound by theory or mechanism. Anytheory or mechanism presented herein is included solely to supplementthe disclosure, and should not be interpreted to impose any limitationon the claims presented hereinbelow. Therefore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation—the invention being defined by the claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 10 <210> SEQ ID NO 1 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1Asp Arg Val Tyr Ile His Pro Phe His Leu 1               5   #                10 <210> SEQ ID NO 2 <211> LENGTH: 8 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Asp Arg Val Tyr Ile His Pro Phe 1               5 <210> SEQ ID NO 3<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 3 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1               5<210> SEQ ID NO 4 <211> LENGTH: 11 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 4Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Me #t 1               5   #                10 <210> SEQ ID NO 5 <211> LENGTH: 3407 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 5cgcgcgtctc cgccgcccgc gtgacttctg cctgcgctcc ttctctgaac gc#tcacttcc     60gaggagacgc cgacgatgaa gacaccgtgg aagattcttc tgggactgct gg#gtgctgct    120gcgcttgtca ccatcatcac cgtgcccgtg gttctgctga acaaaggcac ag#atgatgct    180acagctgaca gtcgcaaaac ttacactcta actgattact taaaaaatac tt#atagactg    240aagttatact ccttaagatg gatttcagat catgaatatc tctacaaaca ag#aaaataat    300atcttggtat tcaatgctga atatggaaac agctcagttt tcttggagaa ca#gtacattt    360gatgagtttg gacattctat caatgattat tcaatatctc ctgatgggca gt#ttattctc    420ttagaataca actacgtgaa gcaatggagg cattcctaca cagcttcata tg#acatttat    480gatttaaata aaaggcagct gattacagaa gagaggattc caaacaacac ac#agtgggtc    540acatggtcac cagtgggtca taaattggca tatgtttgga acaatgacat tt#atgttaaa    600attgaaccaa atttaccaag ttacagaatc acatggacgg ggaaagaaga ta#taatatat    660aatggaataa ctgactgggt ttatgaagag gaagtcttca gtgcctactc tg#ctctgtgg    720tggtctccaa acggcacttt tttagcatat gcccaattta acgacacaga ag#tcccactt    780attgaatact ccttctactc tgatgagtca ctgcagtacc caaagactgt ac#gggttcca    840tatccaaagg caggagctgt gaatccaact gtaaagttct ttgttgtaaa ta#cagactct    900ctcagctcag tcaccaatgc aacttccata caaatcactg ctcctgcttc ta#tgttgata    960ggggatcact acttgtgtga tgtgacatgg gcaacacaag aaagaatttc tt#tgcagtgg   1020ctcaggagga ttcagaacta ttcggtcatg gatatttgtg actatgatga at#ccagtgga   1080agatggaact gcttagtggc acggcaacac attgaaatga gtactactgg ct#gggttgga   1140agatttaggc cttcagaacc tcattttacc cttgatggta atagcttcta ca#agatcatc   1200agcaatgaag aaggttacag acacatttgc tatttccaaa tagataaaaa ag#actgcaca   1260tttattacaa aaggcacctg ggaagtcatc gggatagaag ctctaaccag tg#attatcta   1320tactacatta gtaatgaata taaaggaatg ccaggaggaa ggaatcttta ta#aaatccaa   1380cttattgact atacaaaagt gacatgcctc agttgtgagc tgaatccgga aa#ggtgtcag   1440tactattctg tgtcattcag taaagaggcg aagtattatc agctgagatg tt#ccggtcct   1500ggtctgcccc tctatactct acacagcagc gtgaatgata aagggctgag ag#tcctggaa   1560gacaattcag ctttggataa aatgctgcag aatgtccaga tgccctccaa aa#aactggac   1620ttcattattt tgaatgaaac aaaattttgg tatcagatga tcttgcctcc tc#attttgat   1680aaatccaaga aatatcctct actattagat gtgtatgcag gcccatgtag tc#aaaaagca   1740gacactgtct tcagactgaa ctgggccact taccttgcaa gcacagaaaa ca#ttatagta   1800gctagctttg atggcagagg aagtggttac caaggagata agatcatgca tg#caatcaac   1860agaagactgg gaacatttga agttgaagat caaattgaag cagccagaca at#tttcaaaa   1920atgggatttg tggacaacaa acgaattgca atttggggct ggtcatatgg ag#ggtacgta   1980acctcaatgg tcctgggatc gggaagtggc gtgttcaagt gtggaatagc cg#tggcgcct   2040gtatcccggt gggagtacta tgactcagtg tacacagaac gttacatggg tc#tcccaact   2100ccagaagaca accttgacca ttacagaaat tcaacagtca tgagcagagc tg#aaaatttt   2160aaacaagttg agtacctcct tattcatgga acagcagatg ataacgttca ct#ttcagcag   2220tcagctcaga tctccaaagc cctggtcgat gttggagtgg atttccaggc aa#tgtggtat   2280actgatgaag accatggaat agctagcagc acagcacacc aacatatata ta#cccacatg   2340agccacttca taaaacaatg tttctcttta ccttagcacc tcaaaatacc at#gccattta   2400aagcttatta aaactcattt ttgttttcat tatctcaaaa ctgcactgtc aa#gatgatga   2460tgatctttaa aatacacact caaatcaaga aacttaaggt tacctttgtt cc#caaatttc   2520atacctatca tcttaagtag ggacttctgt cttcacaaca gattattacc tt#acagaagt   2580ttgaattatc cggtcgggtt ttattgttta aaatcatttc tgcatcagct gc#tgaaacaa   2640caaataggaa ttgtttttat ggaggctttg catagattcc ctgagcagga tt#ttaatctt   2700tttctaactg gactggttca aatgttgttc tcttctttaa agggatggca ag#atgtgggc   2760agtgatgtca ctagggcagg gacaggataa gagggattag ggagagaaga ta#gcagggca   2820tggctgggaa cccaagtcca agcataccaa cacgagcagg ctactgtcag ct#cccctcgg   2880agaagagctg ttcaccacga gactggcaca gttttctgag aaagactatt ca#aacagtct   2940caggaaatca aatatcgaaa gcactgactt ctaagtaaac cacagcagtt ga#aagactcc   3000aaagaaatgt aagggaaact gccagcaacg cagcccccag gtgccagtta tg#gctatagg   3060tgctacaaaa acacagcaag ggtgatggga aagcattgta aatgtgcttt ta#aaaaaaaa   3120tactgatgtt cctagtgaaa gaggcagctt gaaactgaga tgtgaacaca tc#agcttgcc   3180ctgttaaaag atgaaaatat ttgtatcaca aatcttaact tgaaggagtc ct#tgcatcaa   3240tttttcttat ttcatttctt tgagtgtctt aattaaaaga atattttaac tt#ccttggac   3300tcattttaaa aaatggaaca taaaatacaa tgttatgtat tattattccc at#tctacata   3360 ctatggaatt tctcccagtc atttaataaa tgtgccttca ttttttc   #              3407 <210> SEQ ID NO 6 <211> LENGTH: 766 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 6Met Lys Thr Pro Trp Lys Ile Leu Leu Gly Le #u Leu Gly Ala Ala Ala1               5    #                10   #                15Leu Val Thr Ile Ile Thr Val Pro Val Val Le #u Leu Asn Lys Gly Thr            20       #            25       #            30Asp Asp Ala Thr Ala Asp Ser Arg Lys Thr Ty #r Thr Leu Thr Asp Tyr        35           #        40           #        45Leu Lys Asn Thr Tyr Arg Leu Lys Leu Tyr Se #r Leu Arg Trp Ile Ser    50               #    55               #    60Asp His Glu Tyr Leu Tyr Lys Gln Glu Asn As #n Ile Leu Val Phe Asn65                   #70                   #75                   #80Ala Glu Tyr Gly Asn Ser Ser Val Phe Leu Gl #u Asn Ser Thr Phe Asp                85   #                90   #                95Glu Phe Gly His Ser Ile Asn Asp Tyr Ser Il #e Ser Pro Asp Gly Gln            100       #           105       #           110Phe Ile Leu Leu Glu Tyr Asn Tyr Val Lys Gl #n Trp Arg His Ser Tyr        115           #       120           #       125Thr Ala Ser Tyr Asp Ile Tyr Asp Leu Asn Ly #s Arg Gln Leu Ile Thr    130               #   135               #   140Glu Glu Arg Ile Pro Asn Asn Thr Gln Trp Va #l Thr Trp Ser Pro Val145                 1 #50                 1 #55                 1 #60Gly His Lys Leu Ala Tyr Val Trp Asn Asn As #p Ile Tyr Val Lys Ile                165   #               170   #               175Glu Pro Asn Leu Pro Ser Tyr Arg Ile Thr Tr #p Thr Gly Lys Glu Asp            180       #           185       #           190Ile Ile Tyr Asn Gly Ile Thr Asp Trp Val Ty #r Glu Glu Glu Val Phe        195           #       200           #       205Ser Ala Tyr Ser Ala Leu Trp Trp Ser Pro As #n Gly Thr Phe Leu Ala    210               #   215               #   220Tyr Ala Gln Phe Asn Asp Thr Glu Val Pro Le #u Ile Glu Tyr Ser Phe225                 2 #30                 2 #35                 2 #40Tyr Ser Asp Glu Ser Leu Gln Tyr Pro Lys Th #r Val Arg Val Pro Tyr                245   #               250   #               255Pro Lys Ala Gly Ala Val Asn Pro Thr Val Ly #s Phe Phe Val Val Asn            260       #           265       #           270Thr Asp Ser Leu Ser Ser Val Thr Asn Ala Th #r Ser Ile Gln Ile Thr        275           #       280           #       285Ala Pro Ala Ser Met Leu Ile Gly Asp His Ty #r Leu Cys Asp Val Thr    290               #   295               #   300Trp Ala Thr Gln Glu Arg Ile Ser Leu Gln Tr #p Leu Arg Arg Ile Gln305                 3 #10                 3 #15                 3 #20Asn Tyr Ser Val Met Asp Ile Cys Asp Tyr As #p Glu Ser Ser Gly Arg                325   #               330   #               335Trp Asn Cys Leu Val Ala Arg Gln His Ile Gl #u Met Ser Thr Thr Gly            340       #           345       #           350Trp Val Gly Arg Phe Arg Pro Ser Glu Pro Hi #s Phe Thr Leu Asp Gly        355           #       360           #       365Asn Ser Phe Tyr Lys Ile Ile Ser Asn Glu Gl #u Gly Tyr Arg His Ile    370               #   375               #   380Cys Tyr Phe Gln Ile Asp Lys Lys Asp Cys Th #r Phe Ile Thr Lys Gly385                 3 #90                 3 #95                 4 #00Thr Trp Glu Val Ile Gly Ile Glu Ala Leu Th #r Ser Asp Tyr Leu Tyr                405   #               410   #               415Tyr Ile Ser Asn Glu Tyr Lys Gly Met Pro Gl #y Gly Arg Asn Leu Tyr            420       #           425       #           430Lys Ile Gln Leu Ile Asp Tyr Thr Lys Val Th #r Cys Leu Ser Cys Glu        435           #       440           #       445Leu Asn Pro Glu Arg Cys Gln Tyr Tyr Ser Va #l Ser Phe Ser Lys Glu    450               #   455               #   460Ala Lys Tyr Tyr Gln Leu Arg Cys Ser Gly Pr #o Gly Leu Pro Leu Tyr465                 4 #70                 4 #75                 4 #80Thr Leu His Ser Ser Val Asn Asp Lys Gly Le #u Arg Val Leu Glu Asp                485   #               490   #               495Asn Ser Ala Leu Asp Lys Met Leu Gln Asn Va #l Gln Met Pro Ser Lys            500       #           505       #           510Lys Leu Asp Phe Ile Ile Leu Asn Glu Thr Ly #s Phe Trp Tyr Gln Met        515           #       520           #       525Ile Leu Pro Pro His Phe Asp Lys Ser Lys Ly #s Tyr Pro Leu Leu Leu    530               #   535               #   540Asp Val Tyr Ala Gly Pro Cys Ser Gln Lys Al #a Asp Thr Val Phe Arg545                 5 #50                 5 #55                 5 #60Leu Asn Trp Ala Thr Tyr Leu Ala Ser Thr Gl #u Asn Ile Ile Val Ala                565   #               570   #               575Ser Phe Asp Gly Arg Gly Ser Gly Tyr Gln Gl #y Asp Lys Ile Met His            580       #           585       #           590Ala Ile Asn Arg Arg Leu Gly Thr Phe Glu Va #l Glu Asp Gln Ile Glu        595           #       600           #       605Ala Ala Arg Gln Phe Ser Lys Met Gly Phe Va #l Asp Asn Lys Arg Ile    610               #   615               #   620Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Va #l Thr Ser Met Val Leu625                 6 #30                 6 #35                 6 #40Gly Ser Gly Ser Gly Val Phe Lys Cys Gly Il #e Ala Val Ala Pro Val                645   #               650   #               655Ser Arg Trp Glu Tyr Tyr Asp Ser Val Tyr Th #r Glu Arg Tyr Met Gly            660       #           665       #           670Leu Pro Thr Pro Glu Asp Asn Leu Asp His Ty #r Arg Asn Ser Thr Val        675           #       680           #       685Met Ser Arg Ala Glu Asn Phe Lys Gln Val Gl #u Tyr Leu Leu Ile His    690               #   695               #   700Gly Thr Ala Asp Asp Asn Val His Phe Gln Gl #n Ser Ala Gln Ile Ser705                 7 #10                 7 #15                 7 #20Lys Ala Leu Val Asp Val Gly Val Asp Phe Gl #n Ala Met Trp Tyr Thr                725   #               730   #               735Asp Glu Asp His Gly Ile Ala Ser Ser Thr Al #a His Gln His Ile Tyr            740       #           745       #           750Thr His Met Ser His Phe Ile Lys Gln Cys Ph #e Ser Leu Pro        755           #       760           #       765<210> SEQ ID NO 7 <211> LENGTH: 2366 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)..(2366) <223> OTHER INFORMATION: n is any nucleotide<400> SEQUENCE: 7gcgnccgctc ccacttcaga ttgaacctaa cgaggtgaca cactcaggag ac#acaggtgt     60ggaaacagac ggcagaatgc ctccaaaggt gacttcagag ctgcttcggc ag#ctgagaca    120agccatgagg aactctgagt atgtgaccga accgatccag gcctacatca tc#ccatcggg    180agatgctcat cagagtgagt atattgctcc atgtgactgt cggcgggctt tt#gtctctgg    240attcgatggc tctgcgggca cagccatcat cacagaagag catgcagcca tg#tggactga    300cgggcgctac tttctccagg ctgccaagca aatggacagc aactggacac tt#atgaagat    360gggtctgaag gacacaccaa ctcaggaaga ctggctggtg agtgtgcttc ct#gaaggatc    420cagggttggt gtggacccct tgatcattcc tacagattat tggaagaaaa tg#gccaaagt    480tctgagaagt gccggccatc acctcattcc tgtcaaggag aacctcgttg ac#aaaatctg    540gacagaccgt cctgagcgcc cttgcaagcc tctcctcaca ctgggcctgg at#tacacagg    600catctcctgg aaggacaagg ttgcagacct tcggttgaaa atggctgaga gg#aacgtcat    660gtggtttgtg gtcactgcct tggatgagat tgcgtggcta tttaatctcc ga#ggatcaga    720tgtggagcac aatccagtat ttttctccta cgcaatcata ggactagaga cg#atcatgct    780cttcattgat ggtgaccgca tagacgcccc cagtgtgaag gagcacctgc tt#cttgactt    840gggtctggaa gccgaataca ggatccaggt gcatccctac aagtccatcc tg#agcgagct    900caaggccctg tgtgctgacc tctccccaag ggagaaggtg tgggtcagtg ac#aaggccag    960ctatgctgtg agcgagacca tccccaagga ccaccgctgc tgtatgcctt ac#acccccat   1020ctgcatcgcc aaagctgtga agaattcagc tgagtcagaa ggcatgaggc cg#gctcacat   1080taaagatgct gttgctctct gtgaactctt taactggctg gagaaagagg tt#cccaaagg   1140tggtgtgaca gagatctcag ctgctgacaa agctgaggag tttcgcaggc aa#caggcaga   1200ctttgtggac ctgagcttcc caacaatttc cagtacggga cccaacggcg cc#atcattca   1260ctacgcgcca gtccctgaga cgaataggac cttgtccctg gatgaggtgt ac#cttattga   1320ctcgggtgct caatacaagg atggcaccac agatgtgacg cggacaatgc at#tttgggac   1380ccctacagcc tacgagaagg aatgcttcac atatgtcctc aagggccaca ta#gctgtgag   1440tgcagccgtt ttcccgactg gaaccaaagg tcaccttctt gactcctttg cc#cgttcagc   1500tttatgggat tcaggcctag attacttgca cgggactgga catggtgttg gg#tctttttt   1560gaatgtccat gagggtcctt gcggcatcag ttacaaaaca ttctctgatg ag#cccttgga   1620ggcaggcatg attgtcactg atgagcccgg gtactatgaa gatggggctt tt#ggaattcg   1680cattgagaat gttgtccttg tggttcctgt gaagaccaag tataatttta at#aaccgggg   1740aagcctgacc tttgaacctc taacattggt tccaattcag accaaaatga ta#gatgtgga   1800ttctcttaca gacaaagagt gcgactggct caacaattac cacctgacct gc#agggatgt   1860gattgggaag gaattgcaga aacagggccg ccaggaagct ctcgagtggc tc#atcagaga   1920gacgcaaccc atctccaaac agcattaata aatacctccc cggttttgtt tt#tgtaaaat   1980gctctggagg aaggaagaaa cgtggcagat ccctgacatc tttccccttt cc#tttccttc   2040ttccctacct ccccttttta ctttagactt taagaagaac agaaaatctt ct#tatcctct   2100ttgatatttt attgcaaaca ctcagtcttt tatgattttt taattgttga ga#acaagcca   2160agaataaaat tgctgcacca gaaggagggt ccctccaaag ttgaacactt gg#tgaaagga   2220agatgccccg acttctttgg ccagtgatgg ggaatcagtg agtgctccat ga#tggtcatg   2280ttccaggtgc tagtacatca ttcatgatca ccttaatgct catgagacta ta#tttatgat   2340 cagtgaataa aaatgtcaga actgtg          #                   #            2366 <210> SEQ ID NO 8<211> LENGTH: 623 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 8 Met Pro Pro Lys Val Thr Ser Glu Leu Leu Ar#g Gln Leu Arg Gln Ala 1               5    #                10  #                15 Met Arg Asn Ser Glu Tyr Val Thr Glu Pro Il#e Gln Ala Tyr Ile Ile             20       #            25      #            30 Pro Ser Gly Asp Ala His Gln Ser Glu Tyr Il#e Ala Pro Cys Asp Cys         35           #        40          #        45 Arg Arg Ala Phe Val Ser Gly Phe Asp Gly Se#r Ala Gly Thr Ala Ile     50               #    55              #    60 Ile Thr Glu Glu His Ala Ala Met Trp Thr As#p Gly Arg Tyr Phe Leu 65                   #70                  #75                   #80 Gln Ala Ala Lys Gln Met Asp Ser Asn Trp Th#r Leu Met Lys Met Gly                 85   #                90  #                95 Leu Lys Asp Thr Pro Thr Gln Glu Asp Trp Le#u Val Ser Val Leu Pro             100       #           105      #           110 Glu Gly Ser Arg Val Gly Val Asp Pro Leu Il#e Ile Pro Thr Asp Tyr         115           #       120          #       125 Trp Lys Lys Met Ala Lys Val Leu Arg Ser Al#a Gly His His Leu Ile     130               #   135              #   140 Pro Val Lys Glu Asn Leu Val Asp Lys Ile Tr#p Thr Asp Arg Pro Glu 145                 1 #50                 1#55                 1 #60 Arg Pro Cys Lys Pro Leu Leu Thr Leu Gly Le#u Asp Tyr Thr Gly Ile                 165   #               170  #               175 Ser Trp Lys Asp Lys Val Ala Asp Leu Arg Le#u Lys Met Ala Glu Arg             180       #           185      #           190 Asn Val Met Trp Phe Val Val Thr Ala Leu As#p Glu Ile Ala Trp Leu         195           #       200          #       205 Phe Asn Leu Arg Gly Ser Asp Val Glu His As#n Pro Val Phe Phe Ser     210               #   215              #   220 Tyr Ala Ile Ile Gly Leu Glu Thr Ile Met Le#u Phe Ile Asp Gly Asp 225                 2 #30                 2#35                 2 #40 Arg Ile Asp Ala Pro Ser Val Lys Glu His Le#u Leu Leu Asp Leu Gly                 245   #               250  #               255 Leu Glu Ala Glu Tyr Arg Ile Gln Val His Pr#o Tyr Lys Ser Ile Leu             260       #           265      #           270 Ser Glu Leu Lys Ala Leu Cys Ala Asp Leu Se#r Pro Arg Glu Lys Val         275           #       280          #       285 Trp Val Ser Asp Lys Ala Ser Tyr Ala Val Se#r Glu Thr Ile Pro Lys     290               #   295              #   300 Asp His Arg Cys Cys Met Pro Tyr Thr Pro Il#e Cys Ile Ala Lys Ala 305                 3 #10                 3#15                 3 #20 Val Lys Asn Ser Ala Glu Ser Glu Gly Met Ar#g Pro Ala His Ile Lys                 325   #               330  #               335 Asp Ala Val Ala Leu Cys Glu Leu Phe Asn Tr#p Leu Glu Lys Glu Val             340       #           345      #           350 Pro Lys Gly Gly Val Thr Glu Ile Ser Ala Al#a Asp Lys Ala Glu Glu         355           #       360          #       365 Phe Arg Arg Gln Gln Ala Asp Phe Val Asp Le#u Ser Phe Pro Thr Ile     370               #   375              #   380 Ser Ser Thr Gly Pro Asn Gly Ala Ile Ile Hi#s Tyr Ala Pro Val Pro 385                 3 #90                 3#95                 4 #00 Glu Thr Asn Arg Thr Leu Ser Leu Asp Glu Va#l Tyr Leu Ile Asp Ser                 405   #               410  #               415 Gly Ala Gln Tyr Lys Asp Gly Thr Thr Asp Va#l Thr Arg Thr Met His             420       #           425      #           430 Phe Gly Thr Pro Thr Ala Tyr Glu Lys Glu Cy#s Phe Thr Tyr Val Leu         435           #       440          #       445 Lys Gly His Ile Ala Val Ser Ala Ala Val Ph#e Pro Thr Gly Thr Lys     450               #   455              #   460 Gly His Leu Leu Asp Ser Phe Ala Arg Ser Al#a Leu Trp Asp Ser Gly 465                 4 #70                 4#75                 4 #80 Leu Asp Tyr Leu His Gly Thr Gly His Gly Va#l Gly Ser Phe Leu Asn                 485   #               490  #               495 Val His Glu Gly Pro Cys Gly Ile Ser Tyr Ly#s Thr Phe Ser Asp Glu             500       #           505      #           510 Pro Leu Glu Ala Gly Met Ile Val Thr Asp Gl#u Pro Gly Tyr Tyr Glu         515           #       520          #       525 Asp Gly Ala Phe Gly Ile Arg Ile Glu Asn Va#l Val Leu Val Val Pro     530               #   535              #   540 Val Lys Thr Lys Tyr Asn Phe Asn Asn Arg Gl#y Ser Leu Thr Phe Glu 545                 5 #50                 5#55                 5 #60 Pro Leu Thr Leu Val Pro Ile Gln Thr Lys Me#t Ile Asp Val Asp Ser                 565   #               570  #               575 Leu Thr Asp Lys Glu Cys Asp Trp Leu Asn As#n Tyr His Leu Thr Cys             580       #           585      #           590 Arg Asp Val Ile Gly Lys Glu Leu Gln Lys Gl#n Gly Arg Gln Glu Ala         595           #       600          #       605 Leu Glu Trp Leu Ile Arg Glu Thr Gln Pro Il#e Ser Lys Gln His     610               #   615               #   620<210> SEQ ID NO 9 <211> LENGTH: 3428 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 9caccctatcc tacactacta ggaacttgca cagtccgcct cgggcagccc aa#agctcctc     60tgcccaccct ggctcccaaa accctccaaa acaaaagacc agaaaagcac tc#tccaccca    120gcagccaaac gcctccttct tgacgccagc ccccaccctc tgtctgctcg ag#cccaggaa    180aggcctgaag gaacaggccg gggaaggagc cctccctctc tcccttgtcc ct#ccatccac    240ccagcgccgg catctggaga ccctatggcc cgggctcact ggggctgctg cc#cctggctg    300gtcctcctct gtgcttgtgc ctggggccac acaaagccac tggaccttgg ag#ggcaggat    360gtgagaaatt gttccaccaa ccccccttac cttccagtta ctgtggtcaa ta#ccacaatg    420tcactcacag ccctccgcca gcagatgcag acccagaatc tctcagccta ca#tcatccca    480ggcacagatg ctcacatgaa cgagtacatc ggccaacatg acgagaggcg tg#cgtggatt    540acaggcttta cagggtctgc aggaactgca gtggtgacta tgaagaaagc ag#ctgtctgg    600accgacagtc gctactggac tcaggctgag cggcaaatgg actgtaattg gg#agctccat    660aaggaagttg gcaccactcc tattgtcacc tggctcctca ccgagattcc cg#ctggaggg    720cgtgtgggtt ttgacccctt cctcttgtcc attgacacct gggagagtta tg#atctggcc    780ctccaaggct ctaacagaca gctggtgtcc atcacaacca atcttgtgga cc#tggtatgg    840ggatcagaga ggccaccggt tccaaatcaa cccatttatg ccctgcagga gg#cattcaca    900gggagcactt ggcaggagaa agtatctggc gtccgaagcc agatgcagaa gc#atcaaaag    960gtcccgactg ccgtccttct gtcggcgctt gaggagacgg cctggctctt ca#accttcga   1020gccagtgaca tcccctataa ccccttcttc tattcctaca cgctgctcac ag#actcttct   1080attaggttgt ttgcaaacaa gagtcgcttt agctccgaaa ccttgagcta tc#tgaactcc   1140agttgcacag gccccatgtg tgtgcaaatc gaggattaca gccaagttcg tg#acagcatc   1200caggcctact cattgggaga tgtgaggatc tggattggga ccagctatac ca#tgtatggg   1260atctatgaaa tgataccaag ggagaaactc gtgacagaca cctactcccc ag#tgatgatg   1320accaaggcag tgaagaacag caaggagcag gccctcctca aggccagcca cg#tgcgggac   1380gctgtggctg tgatccggta cttggtctgg ctggagaaga acgtgcccaa ag#gcacagtg   1440gatgagtttt cgggggcaga gatcgtggac aagttccgag gagaagaaca gt#tctcctcc   1500ggacccagtt ttgaaaccat ctctgctagt ggtttgaatg ctgccctggc cc#actacagc   1560ccgaccaagg agctgaaccg caagctgtcc tcagatgaga tgtacctgct gg#actctggg   1620gggcagtact gggacgggac cacagacatc accagaacag tccactgggg ca#ccccctct   1680gcctttcaga aggaggcata tacccgtgtg ctgataggaa atattgacct gt#ccaggctc   1740atctttcccg ctgctacatc agggcgaatg gtggaggcct ttgcccgcag ag#ccttgtgg   1800gatgctggtc tcaattatgg tcatgggaca ggccacggca ttggcaactt cc#tgtgtgtg   1860catgagtggc cagtgggatt ccagtccaac aacatcgcta tggccaaggg ca#tgttcact   1920tccattgaac ctggttacta taaggatgga gaatttggga tccgtctcga ag#atgtggct   1980ctcgtggtag aagcaaagac caagtaccca ggggagctac ctgaccttgt gg#tatcattt   2040gtgccctatg accggaacct catcgatgtc agcctgctgt ctcccgagca tc#tccagtac   2100ctgaatcgct actaccagac catccgggag aaggtgggtc cagagctgca ga#ggcgccag   2160ctactagagg agttcgagtg gcttcaacag cacacagagc ccctggccgc ca#gggcccca   2220gacaccgcct cctgggcctc tgtgttagtg gtctccaccc ttgccatcct tg#gctggagt   2280gtctagaggc tccagactct cctgttaacc ctccatctag atggggggct cc#cttgctta   2340gctcccctca ccctgcactg aacatacccc aagagcccct gctggcccat tg#cctagaaa   2400cctttgcatt catcctcctt ctccaagacc tatggagaag gtcccaggcc cc#aggaaaca   2460cagggcttct tggccccaga tggcacctcc ctgcaccccg gggttgtata cc#acaccctg   2520ggcccctaat cccaggcccc gaaataggaa agccagctag tctcttctct tc#tgtgatct   2580cagtaggcct aacctataac ctaacacaga ctgctacagc tgctcccctc cc#gccaaaca   2640aagccccaag aaaacaatgc ccctaccacc caagggtgcc atggtcccgg ga#aaacccaa   2700cctgtcaccg cgtgttgggc gtaaccagaa ctgttccccc ccaccagggc tt#aaaaatcg   2760cccccacttt ttaaccatcg tccattaacc acctggtggg catagccaga gc#tgttcgaa   2820cccagccagg gatgaaaaat caacccccga catggaaccc atgattccta aa#cccggggt   2880aggttccatg ccaagtaaca gcagagggag ttaagccata ggaatttggc tg#tggagtaa   2940gagggaatgc ggtgaggcag tgtggaatat gaccctacca gaggttggag aa#caaacttg   3000ggcagccgga acccgtcact attttagatt cctggcattc gaggagccct tt#gaactttc   3060caaagtgcag ccacagctac aatgctgtta aatcctccca catttcttgg at#gccccttc   3120accttgtgtg gacagtgtct ggtttcccca ttttacagac aggaaaactg ag#cttcagac   3180agggggtggg ctttgcctaa ggacacacaa atttggttgg gagttgatgg gg#ccagatga   3240gccagcattc cagctgtttc acccttcagc aacatgcaga gtccctgagc cc#acctccca   3300gccctctcct cattctctga acccactgtg gtgagaagaa tttgctccgg cc#aaattggc   3360cgttagccac ctgggtccac atcctgctaa gacgtttaaa acagcctaac aa#agacactt   3420 gcctgtgg                 #                  #                   #        3428 <210> SEQ ID NO 10 <211> LENGTH: 493<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 10Val Ser Ile Thr Thr Asn Leu Val Asp Leu Va #l Trp Gly Ser Glu Arg1               5    #                10   #                15Pro Pro Val Pro Asn Gln Pro Ile Tyr Ala Le #u Gln Glu Ala Phe Thr            20       #            25       #            30Gly Ser Thr Trp Gln Glu Lys Val Ser Gly Va #l Arg Ser Gln Met Gln        35           #        40           #        45Lys His Gln Lys Val Pro Thr Ala Val Leu Le #u Ser Ala Leu Glu Glu    50               #    55               #    60Thr Ala Trp Leu Phe Asn Leu Arg Ala Ser As #p Ile Pro Tyr Asn Pro65                   #70                   #75                   #80Phe Phe Tyr Ser Tyr Thr Leu Leu Thr Asp Se #r Ser Ile Arg Leu Phe                85   #                90   #                95Ala Asn Lys Ser Arg Phe Ser Ser Glu Thr Le #u Ser Tyr Leu Asn Ser            100       #           105       #           110Ser Cys Thr Gly Pro Met Cys Val Gln Ile Gl #u Asp Tyr Ser Gln Val        115           #       120           #       125Arg Asp Ser Ile Gln Ala Tyr Ser Leu Gly As #p Val Arg Ile Trp Ile    130               #   135               #   140Gly Thr Ser Tyr Thr Met Tyr Gly Ile Tyr Gl #u Met Ile Pro Arg Glu145                 1 #50                 1 #55                 1 #60Lys Leu Val Thr Asp Thr Tyr Ser Pro Val Me #t Met Thr Lys Ala Val                165   #               170   #               175Lys Asn Ser Lys Glu Gln Ala Leu Leu Lys Al #a Ser His Val Arg Asp            180       #           185       #           190Ala Val Ala Val Ile Arg Tyr Leu Val Trp Le #u Glu Lys Asn Val Pro        195           #       200           #       205Lys Gly Thr Val Asp Glu Phe Ser Gly Ala Gl #u Ile Val Asp Lys Phe    210               #   215               #   220Arg Gly Glu Glu Gln Phe Ser Ser Gly Pro Se #r Phe Glu Thr Ile Ser225                 2 #30                 2 #35                 2 #40Ala Ser Gly Leu Asn Ala Ala Leu Ala His Ty #r Ser Pro Thr Lys Glu                245   #               250   #               255Leu Asn Arg Lys Leu Ser Ser Asp Glu Met Ty #r Leu Leu Asp Ser Gly            260       #           265       #           270Gly Gln Tyr Trp Asp Gly Thr Thr Asp Ile Th #r Arg Thr Val His Trp        275           #       280           #       285Gly Thr Pro Ser Ala Phe Gln Lys Glu Ala Ty #r Thr Arg Val Leu Ile    290               #   295               #   300Gly Asn Ile Asp Leu Ser Arg Leu Ile Phe Pr #o Ala Ala Thr Ser Gly305                 3 #10                 3 #15                 3 #20Arg Met Val Glu Ala Phe Ala Arg Arg Ala Le #u Trp Asp Ala Gly Leu                325   #               330   #               335Asn Tyr Gly His Gly Thr Gly His Gly Ile Gl #y Asn Phe Leu Cys Val            340       #           345       #           350His Glu Trp Pro Val Gly Phe Gln Ser Asn As #n Ile Ala Met Ala Lys        355           #       360           #       365Gly Met Phe Thr Ser Ile Glu Pro Gly Tyr Ty #r Lys Asp Gly Glu Phe    370               #   375               #   380Gly Ile Arg Leu Glu Asp Val Ala Leu Val Va #l Glu Ala Lys Thr Lys385                 3 #90                 3 #95                 4 #00Tyr Pro Gly Glu Leu Pro Asp Leu Val Val Se #r Phe Val Pro Tyr Asp                405   #               410   #               415Arg Asn Leu Ile Asp Val Ser Leu Leu Ser Pr #o Glu His Leu Gln Tyr            420       #           425       #           430Leu Asn Arg Tyr Tyr Gln Thr Ile Arg Glu Ly #s Val Gly Pro Glu Leu        435           #       440           #       445Gln Arg Arg Gln Leu Leu Glu Glu Phe Glu Tr #p Leu Gln Gln His Thr    450               #   455               #   460Glu Pro Leu Ala Ala Arg Ala Pro Asp Thr Al #a Ser Trp Ala Ser Val465                 4 #70                 4 #75                 4 #80Leu Val Val Ser Thr Leu Ala Ile Leu Gly Tr #p Ser Val                485   #               490

What is claimed is:
 1. A method of identifying a subject that issusceptible to developing an angioedemic condition during a course oftreatment comprising administering one of an ACE inhibitor and avasopeptidase inhibitor, comprising: (a) providing a biological samplefrom a subject; (b) determining a dipeptidyl peptidase IV activity inthe biological sample; and (c) comparing a dipeptidyl peptidase IVactivity in the biological sample to a standard dipeptidyl peptidase IVactivity, wherein a 10% or more reduction in the sample activitycompared to the standard indicates that the subject is susceptible todeveloping an angioedema during a course of treatment comprisingadministering one of an ACE inhibitor and a vasopeptidase inhibitor. 2.The method of claim 1, wherein the vasopeptidase inhibitor comprises anangiotensin-converting enzyme inhibitor.
 3. The method of claim 1,wherein the vasopeptidase inhibitor comprises a neutral endopeptidaseinhibitor.
 4. The method of claim 1, wherein the subject is a human. 5.The method of claim 1, wherein a 20% or more reduction in the dipeptidylpeptidase IV activity in the biological sample, as compared to thestandard dipeptidyl peptidase IV activity indicates that the subject issusceptible to developing an angioedema during a course of treatmentcomprising administering one of an ACE inhibitor and a vasopeptidaseinhibitor.
 6. The method of claim 1, wherein a 30% or more reduction inthe dipeptidyl peptidase IV activity in the biological sample, ascompared to the standard dipeptidyl peptidase IV activity indicates thatthe subject is susceptible to developing an angioedema during a courseof treatment comprising administering one of an ACE inhibitor and avasopeptidase inhibitor.
 7. A method of determining contraindication foradministration of one of an ACE inhibitor and a vasopeptidase inhibitorto an individual, comprising: (a) providing a biological sample from asubject; (b) determining a dipeptidyl peptidase IV activity in thebiological sample; and (c) comparing a dipeptidyl peptidase IV activityin the biological sample to a standard dipeptidyl peptidase IV activityrange, wherein administration of the vasopeptidase inhibitor iscontraindicated when the dipeptidyl peptidase IV activity in thebiological sample is in a range that is below the standard dipeptidylpeptidase IV activity range by a significant difference and thesignificance is greater than or equal to a 95% confidence interval. 8.The method of claim 7, wherein the vasopeptidase inhibitor is anangiotensin-converting enzyme inhibitor.
 9. The method of claim 7,wherein the vasopeptidase inhibitor is a neutral endopeptidaseinhibitor.